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B Sites in Mouse CD95 Ligand (Fas Ligand) Promoter: Functional Analysis in T Cell Hybridoma1
Departments of
*
Pathology and Laboratory Medicine,
Medicine, and
Microbiology, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B during T
cell activation is a critical event for FasL gene activation. In the
present study we have identified two NF-B sites (designated
FasL-B1 and FasL-B2) on the promoter (
700 bp) of FasL. The
NF-B sites were identified by electrophoretic mobility shift assay.
Transient transfection reporter analyses showed that the FasL promoter
activity was comparable between a construct that contains both sites
and a shorter construct (433 bp) that contains only the FasL-B1
site. Furthermore, elimination of FasL-B1 by site-directed
mutagenesis significantly inhibited FasL promoter activity. These
observations provide strong evidence that NF-B directly binds to the
FasL-B1 site and up-regulates FasL gene
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (10). In
unactivated cells, NF-B forms a complex with IB (a family of
inhibitors of B), which prevents the migration of NF-B to the
nucleus. Upon activation, IB is phosphorylated, ubiquitinated, and
then degraded by the protease in the proteasome, thereby allowing
nuclear translocation of NF-B (10). The activity of the proteasome
can be specifically inhibited by the bacterial metabolite lactacystin
(11). We have previously shown that lactacystin inhibits IBß
degradation, NF-B (p50/p65 and p50/p50) translocation into the
nucleus, and FasL gene activation (8, 9). Furthermore, Ivanov et al.
have independently demonstrated that treatment with antisense p65
inhibits the nuclear translocation of NF-B and the surface
expression of FasL (12). These studies indicate that NF-B is an
important transcription factor for FasL gene expression. However, they
do not address the question of whether NF-B directly or indirectly
regulates FasL gene expression.
In this study we have identified two NF-B sites in the mouse FasL
promoter. By using a transient transfection reporter assay with
constructs containing different lengths of the promoter, we found that
having one of the two NF-B binding sites is sufficient for FasL gene
expression. Furthermore, elimination of this NF-B site by
site-directed mutagenesis significantly impaired FasL promoter function
during T cell activation. Therefore, our study demonstrates that
NF-B directly regulates FasL gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and characterization of the 5D5 T cell hybridoma
have been described previously (13). The anti-CD3 mAb (145-2C11)
was generated in SCID mice and purified from ascites by protein A
column chromatography. Oligonucleotides bearing potential NF-B sites
were deduced from mouse FasL promoter sequence (see below) and
synthesized (Integrated DNA Technology, Coralville, IA). A mutant form
of the NF-B site was also synthesized. The sequences of these
oligonucleotides are as follows: FasL-B1 (-138 to -128:
gagaaAGGTGTTTCCCttgac), FasL-B2 (-441 to -431:
tcctTGGTCTTTTCCCagtgt), and FasL-B1 mutant
(gagaaAGGTGTTTAAAttgac). The underlined nucleotides
indicate the mutated residues. For site-directed mutagenesis, two
single-stranded oligonucleotides complementary to each other were
synthesized and purified (see below for the sequence). The NF-B
probe for the -chain enhancer (14) (agttgaGGGGACTTTCCcaggc) was
obtained from Promega (Madison, WI). Radioactive
[
-32P]ATP was purchased from New England Nuclear
(Boston, MA). All Abs for supershift assay were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). The Quick-Change Site-Directed
Mutagenesis Kit (catalogue no. 200518) was purchased from Stratagene
(La Jolla, CA). The Qiagen Plasmid Maxiprep Kit was purchased
(catalogue no. 12162) from Qiagen (Santa Clarita, CA). The luciferase
detection kit (catalogue no. 1500) was purchased from Promega.
Cloning of mouse FasL promoter
Employing a series of adaptor-ligated genomic libraries as templates, PCR was used to walk upstream from the 5' end of the mouse FasL cDNA sequence (1). By this method, approximately 700 bp of sequence was obtained (GenBank accession no. AF045739) and was confirmed to be contiguous with the 5' end of the mouse FasL cDNA. Sequence analysis indicated marked similarity to the structure of the human FasL promoter, including the position of the TATA box (15). Constructs containing varying lengths of the mouse FasL promoter positioned upstream from a luciferase reporter gene were then generated for transient transfection studies. The 3' end of each promoter fragment was the first nucleotide upstream from the translational start site.
Nuclear extraction and EMSA
Preparation of nuclear extracts, labeling of oligonucleotides, EMSA, and Ab-mediated supershift assays were conducted as previously described (8, 9). Nuclear extracts were prepared from 5D5 hybridoma T cells at various times after activation with plate-bound anti-CD3 mAb.
Site-directed mutagenesis
Site-direct mutagenesis was conducted according to the manufacturer’s instructions. Briefly, p433, a nonlinearized construct that contains the first 433 bp (upstream of the translational start site) of FasL promoter, was used as a template. No further subcloning was required. A pair of 35-mer oligonucleotides with the designed mutations was synthesized (5'-caggagaaaggtgtttaaattgactgcggaaacct-3' and 5'-aggtttccgcagtcaatttaaacacctttctcctg-3') and used as primers (underlined nucleotides indicate the positions of the mutations). The template DNA and two primers were mixed with the 10x reaction buffer and pfu DNA polymerase that were provided with the kit (temperature cycling reaction: one cycle of 95°C for 30 s, and 12 cycles of 95°C for 30 s, 55°C for 1 min, and 68°C for 13.5 min). After the temperature cycling, DpnI restriction enzyme was added to the reaction mixture to digest the template DNA for 1 h at 37°C. After digestion, bacterial transformation was performed using Epicurian coli XL1-Blue competent cells. Transformed cells were plated on Luria Bertoni broth-agar plate containing ampicillin and were grown overnight at 37°C. A colony was picked and allowed to grow overnight. The plasmid was purified using the Qiagen Plasmid Maxiprep Kit. The mutation was verified by DNA sequencing (data not shown).
Transient transfection and luciferase assay
5D5 cells were transfected with various constructs via electroporation. Typically, 20 x 106 cells were suspended in DMEM without any supplement in 300 µl. The cells were mixed with 6.5 µg of a FasL-luciferase reporter construct together with 3 µg of CMV-pEGFP1 plasmid (provided by Dr. G. Vigilianti, Department of Microbiology, Boston University School of Medicine, Boston, MA), and the mixtures were placed on ice for 10 min. The CMV-pEGFP1 plasmid was used to normalize the transfection efficiency of each sample. The cells were electroporated using a Bio-Rad Gene Pulser at 240 V and 960 µF, then put back into DMEM containing 10% FCS and supplements. They were cultured for 8 h at 37°C, at which point the cells were harvested, purified by Ficoll-Hypaque gradient centrifugation, and counted. A small portion was used for flow cytometric analysis to determine the transfection efficiency. The remaining cells were divided into two groups. One group was cultured in anti-CD3-coated wells (6 µg/ml/well of a 24-well plate, 0.35 x 106 cells/well), and the other group was cultured in uncoated wells. After 16 h, cells were harvested, counted, and lysed with the buffer provided with Promega’s luciferase detection kit (catalogue no. 1500). Lysed samples were mixed with luciferase substrate, and the activity was measured using a luminometer (Turner TD-20e model, Promega). Values from the promoterless pGL3 basic treated under identical conditions were subtracted, and then the fold increase was calculated according to the formula: luciferase value obtained from anti-CD3-coated wells/luciferase value obtained from uncoated wells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B sites
Upon examining the first 700 bases of FasL promoter sequence, two
potential NF-B sites were identified based on the consensus NF-B
sequence. The first potential site was found between -138 to -128,
and the second site was located between -441 to -431 (see
Materials and Methods for the sequence, the numbers are in
reference to the translational start site). We have named the first
site FasL-B1, and the second site FasL-B2. To determine whether
these potential sites can be bound by NF-B, we synthesized two
double-stranded oligonucleotides (21 mer) encompassing the two sites
for EMSA analysis. Labeled probes were incubated with nuclear
extracts from either unactivated or anti-CD3-activated (1.5 h) 5D5
hybridoma T cells. The NF-B probe (22 mer) from the -chain
enhancer region (named B) was included as a positive control. As
reported in previous studies using the control B probe, nuclear
extract from unactivated 5D5 contained basal levels of factors that
bind the probe (8, 9) (Fig. 1
). Upon
anti-CD3 activation, a new band was detected with the activated
nuclear extract. This highly inducible protein was identified to be the
p50/p65 heterodimer of the NF-B family (8, 9). A small increase in
binding was also observed for the second band corresponding to the
p50/p50 homodimer of the NF-B family (8, 9). Although different
binding patterns were observed for FasL-B1 and FasL-B2 probes,
both probes formed a new band with the extract of activated 5D5 cells
(indicated by the upper arrow). This anti-CD3-induced band
comigrated with the p50/p65 heterodimer that bound the B probe (Fig. 1).
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B1 and
FasL-B2 was mediated by p50/p65 heterodimer, Ab-mediated supershift
assays were conducted. When FasL-B1 was used as a probe,
anti-p50 shifted a band corresponding to the p50/50 homodimer
(lower arrow, Fig. 2A), and it
also partially shifted the anti-CD3-induced band. Anti-p65 Ab
strongly inhibited the formation of the anti-CD3-induced band
(upper arrow, Fig. 2A). Moreover, when anti-p50 and
anti-p65 Abs were added together, both bands were displaced. Under
identical conditions, normal rabbit Ig and anti-p52 did not inhibit
the binding. Similarly, anti-c-Rel did not inhibit the binding
significantly. These data demonstrated that FasL-B1 is a NF-B
site that can be bound by the p50/p65 heterodimer present in the
nuclear extract of anti-CD3-activated 5D5 cells. Also, this site
can be bound by the p50/p50 homodimer present in the nuclear extracts
of both unactivated and activated T cells. The same results were
obtained when FasL-B2 probe was used under the same assay condition
(Fig. 2B).
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To determine whether NF-B recognition of the FasL-B1 and
FasL-B2 sites plays a role in FasL gene expression, the relative
activities of reporter genes containing one or more of these sites were
determined. We made one construct containing only the FasL-B1 site
(p433) and another construct that contained both sites (p700). The two
promoter constructs were cloned into the upstream of the luciferase
gene in pGL3 basic. We then transiently transfected 5D5 hybridoma cells
with the pGL3 basic, p433, or p700 construct. To normalize transfection
efficiency, the CMV-pEGFP1 plasmid was cotransfected with each sample.
The percentage of cells expressing the green fluorescent protein
following transfection was between 10 and 13%, and there was no
significant difference in transfection efficiency between various
sample preparations. After the transfection, each group of cells was
divided into two parts. One part was placed into anti-CD3-coated
wells, and the other half was put into uncoated wells. They were
cultured for 16 h, at which point they were collected and lysed,
and then the lysates were measured for luciferase activity. As shown in
Figure 3A, both p433 and p700
constructs showed comparable levels of activity. The fold increases in
luciferase activity upon treatment with anti-CD3 was 5.6 ±
1.2 and 5.4 ± 0.7 for the p700 and p433 constructs, respectively.
The data suggest that sufficient FasL promoter activity can be obtained
even in the absence of the FasL-B2 (-441 to -431) site. Therefore,
we decided to focus on the FasL-B1 site to determine whether NF-B
directly regulates FasL gene expression through binding of FasL-B1
site.
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B). While the p433 construct showed a strong
response upon anti-CD3 stimulation (5.6 ± 0.9-fold increase),
the response of the mutant construct was reduced to 46% of that
activity (2.6 ± 0.5-fold increase). This reduction in luciferase
activity seen with the mutant construct was significant
(p < 0.001; n = 16). To make
sure that the mutation had prevented NF-B from binding to the site,
we synthesized a mutant form of the FasL-B1 oligonucleotide (21 mer)
for EMSA analysis. The unlabeled mutant oligonucleotide was used as a
cold target competitor against 32P-labeled wild-type
FasL-B1 probe. Unlabeled B and FasL-B1 were included as
controls. As shown in Figure 4, both B
and FasL-B1, but not the mutant oligonucleotide, competed
efficiently. Furthermore, when the 32P-labeled mutant was
mixed with nuclear extracts from either unactivated or the
anti-CD3-activated cells, binding by the p50/p50 homodimer and the
p50/p65 heterodimer was not observed (the last three lanes in Fig. 4).
The data indicate that the mutation efficiently eliminated the NF-B
binding site in the FasL-B1 sequence. Taken together with the
transfection analyses, these data demonstrate that the FasL-B1 site
is directly involved in FasL gene expression during T cell activation.
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B1 site is bound by different NF-B in 5D5 at
different times following activation
Because the transfection experiments were assayed at 16 h
after activation, we conducted EMSA analysis to determine whether there
are changes in the Rel family members capable of binding the FasL-B1
probe. Nuclear extracts obtained 4 and 16 h after the activation
of 5D5 cells were compared. The results shown in Figure 5 indicate that at 4 h
postactivation, p50/p65 was still the predominant form of the inducible
Rel family members, similar to the nuclear extract obtained at 1.5
h after activation (see Fig. 2A). However, a low level of
p50/c-Rel binding was detected with the 4-h extract. Moreover, at
16 h postactivation, p50/c-Rel became the major inducible
heterodimer, as indicated by the disappearance of the major inducible
band in the presence of anti-c-Rel Ab. Binding of this probe by
RelB was not detected at any time point. These observations indicate
that there is a change in the inducible Rel family members capable of
binding to the FasL-B1 site and regulating FasL gene expression
during the course of the 16-h activation period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding sites
in the FasL promoter region, and we have demonstrated that one of these
B sites directly contributes to FasL gene expression. The results
presented confirm and extend our previous observations, which
demonstrated that lactacystin blocks NF-B nuclear translocation and
inhibits FasL gene expression as well as activation-induced cell death
and FasL-mediated cytotoxicity (8, 9). Although both sites bind to
NF-B, only one of them (FasL-B1) is sufficient to activate the
FasL gene. The FasL promoter activity of the p433 construct that
contains this site was comparable to that of the construct (p700) that
contains both sites. Moreover, elimination of the FasL-B1site by
site-directed mutagenesis inhibited 54% of the promoter activity
relative to that of the wild-type control, providing strong evidence
that this FasL-B1 site directly regulates FasL gene expression.
The observation that the mutant construct expressed a modest promoter
activity suggests that either there are additional B sites in this
promoter or there are other factors involved in FasL gene regulation.
We tested two additional potential B sites identified based on their
sequence homology to consensus B sequence. The segment from -231 to
-240 contains an NF-AT site that is critical for FasL gene expression
in Jurkat cells (16, 17). However, the antisense, TGGAAGTTTCC, can be a
potential B site. The second potential site we tested was between
-365 and -357 (GGGGTATCC). We could not detect anti-CD3-inducible
NF-B binding when these sites were used as probes in EMSA analyses
(data not shown). However, it remains possible that there are other
NF-B sites present in this promoter. On the other hand, there is a
good evidence implicating NF-AT family members in FasL gene expression,
perhaps in cooperation with the binding of NF-B to the FasL-B1
site that we have identified (16, 17). Both NF-B and NF-AT have been
shown to participate in the regulation of IL-2, which, like FasL, is
preferentially expressed in CD4+ Th1 cells (18, 19).
Latinis et al. have reported that the two NF-AT sites they have
discovered are not important in Sertoli cells, which constitutively
express FasL (20), suggesting again that transcription factors other
than NF-AT are important in FasL gene expression in these cells (16, 17). Studies are in progress to determine whether the FasL-B1 mutant
is functional in Sertoli cells.
The possibility remains that there are additional transcription factors
capable of regulating FasL gene expression during T cell activation. In
this regard, we have identified a segment of the FasL promoter (-194
to -165) that binds SP-1 and at least two other anti-CD3-inducible
nuclear factors. These factors do not appear to be NF-B or NF-AT,
based on cold target competition and Ab-mediated supershift assays. In
addition, one transcription factor could be shifted with anti-Egr-1
Ab, and the other transcription factor could be shifted with
anti-c-Jun Ab (K. Matsui, unpublished observation). Egr-1
has been shown to cooperate with NF-AT for optimal IL-2 promoter
activity (21). It also cooperates with SP-1 for optimal IL-2Rß
promoter activity and with p65 (RelA) for NF-B1 (p105) promoter
activity during T cell activation (22, 23). Experiments are ongoing to
determine whether these transcription factors work in concert with the
anti-CD3-inducible NF-B for optimal expression of FasL promoter
activity.
The observation that the FasL-B2 site is not required for FasL gene
activation in our transient transfection assay does not necessarily
mean that it does not play a role in the activation of the endogenous
FasL gene. It is possible that the transient transfection system has
saturated the effects of NF-B such that binding of both sites does
not produce more activation than binding of Fas-B1 alone. Indeed,
our deletion mutant analysis showed a small but statistically
insignificant contribution of FasL-B2 to FasL promoter activity
(Fig. 3A). A 25% reduction of FasL promoter activity was
obtained in a preliminary study using p700 construct in which the
FasL-B2 was mutated. A study is in progress to determine the effect
of mutating the FasL-B1 site in the p700 construct. If this
construct maintains FasL promoter activity, then it may suggest that
the presence of two functional NF-B sites may serve as a mechanism
safe-guarding the FasL induction system. It should be emphasized that
by using the p433 construct to exclude the FasL-B2 site, we were
able to firmly establish that the FasL-B1 site is an important
element in regulating FasL gene expression.
It is well established that the IL-2 promoter contains NF-B sites,
and it has been shown that the critical NF-B member is c-Rel. IL-2
production induced upon mitogen activation is blocked in T cells from
c-rel knockout mice (24). Moreover, pentoxifylline, a drug
that specifically inhibits c-Rel but not NF-AT, has been reported to
inhibit IL-2 gene expression of peripheral T cells (25). Interestingly,
the same compound also inhibits FasL expression by T cell hybridomas
(S.-T.J., unpublished observations). Moreover, c-Rel expression
requires de novo RNA synthesis, and its appearance in the nucleus
occurs after the translocation of p50/p65 (25). Binding of FasL-B1
by c-Rel was not observed using nuclear extracts from cells activated
for 1.5 h (Fig. 2). However, binding of FasL-B1 by c-Rel was
observed with nuclear extracts from 5D5 cells that had been activated
for 4 and 16 h. We did not detect RelB binding to FasL-B1
probe. Perhaps, FasL promoter initially uses p50/p65 to start the
transcription, then at a later time point it uses c-Rel to maintain the
expression of FasL gene. This interpretation is consistent with the
persistent expression of FasL mRNA and FasL cytotoxicity during the
16 h of activation of 5D5 cells (13).
In contrast to the B probe, FasL-B1 probe was strongly bound by
the p50/p50 homodimer present in the unactivated nuclear extract of 5D5
cells (Fig. 1). Because the p50/p50 homodimer lacks the ability to
activate genes, this may suggest that these sites are occupied by the
p50/p50 homodimers in unactivated 5D5 cells but are unable to express
promoter activity. Upon activation, the p50/p65 heterodimer (or
p50/c-Rel) translocates into the nucleus and bind the FasL-B1 site
by competing off the p50/p50 homodimer. This, in turn, enables the
p50/p65 and p50/c-Rel to activate FasL promoter activity. This
interpretation is consistent with our data showing the continuous
presence of the inducible Rel members capable of activating FasL gene
during prolonged anti-CD3 stimulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S.-T. Ju, K-508, 71 E. Concord Street, Boston University School of Medicine, Boston, MA 02118.
3 Abbreviations used in this paper: FasL, Fas ligand; EMSA, electrophoretic mobility shift assay.
Received for publication April 6, 1998. Accepted for publication June 1, 1998.
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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. Y. Hong, J. H. Park, K. H. Seo, J.-M. Kim, S. Y. Im, J. W. Lee, H.-S. Choi, and K. Lee Expression of MIS in the Testis Is Downregulated by Tumor Necrosis Factor Alpha through the Negative Regulation of SF-1 Transactivation by NF-{kappa}B Mol. Cell. Biol., September 1, 2003; 23(17): 6000 - 6012. [Abstract] [Full Text] [PDF]
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P. Kuenzi, P. Schneider, and D. A. E. Dobbelaere Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas Ligand-Induced Apoptosis J. Immunol., August 1, 2003; 171(3): 1224 - 1231. [Abstract] [Full Text] [PDF]
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 J. Rajakangas, S. Basu, I. Salminen, and M. Mutanen Adenoma Growth Stimulation by the trans-10, cis-12 Isomer of Conjugated Linoleic Acid (CLA) Is Associated with Changes in Mucosal NF-{kappa}B and Cyclin D1 Protein Levels in the Min Mouse J. Nutr., June 1, 2003; 133(6): 1943 - 1948. [Abstract] [Full Text] [PDF]
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I. Muller, S. M. Pfister, U. Grohs, J. Zweigner, R. Handgretinger, D. Niethammer, and G. Bruchelt Receptor Activator of Nuclear Factor {kappa}B Ligand Plays a Nonredundant Role in Doxorubicin-induced Apoptosis Cancer Res., April 15, 2003; 63(8): 1772 - 1775. [Abstract] [Full Text] [PDF]
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K. Roessner, J. Wolfe, C. Shi, L. H. Sigal, S. Huber, and R. C. Budd High Expression of Fas Ligand by Synovial Fluid-Derived {gamma}{delta} T Cells in Lyme Arthritis J. Immunol., March 1, 2003; 170(5): 2702 - 2710. [Abstract] [Full Text] [PDF]
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Y. Yang, B. Dong, P. R. Mittelstadt, H. Xiao, and J. D. Ashwell HIV Tat Binds Egr Proteins and Enhances Egr-dependent Transactivation of the Fas Ligand Promoter J. Biol. Chem., May 24, 2002; 277(22): 19482 - 19487. [Abstract] [Full Text] [PDF]
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W. Ise, M. Totsuka, Y. Sogawa, A. Ametani, S. Hachimura, T. Sato, Y. Kumagai, S. Habu, and S. Kaminogawa Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen J. Immunol., April 1, 2002; 168(7): 3242 - 3250. [Abstract] [Full Text] [PDF]
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N. Ishibashi, O. Prokopenko, K. R. Reuhl, and O. Mirochnitchenko Inflammatory Response and Glutathione Peroxidase in a Model of Stroke J. Immunol., February 15, 2002; 168(4): 1926 - 1933. [Abstract] [Full Text] [PDF]
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 S. Devadas, L. Zaritskaya, S. G. Rhee, L. Oberley, and M. S. Williams Discrete Generation of Superoxide and Hydrogen Peroxide by T Cell Receptor Stimulation: Selective Regulation of Mitogen-Activated Protein Kinase Activation and Fas Ligand Expression J. Exp. Med., January 7, 2002; 195(1): 59 - 70. [Abstract] [Full Text] [PDF]
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 V. Pentikainen, L. Suomalainen, K. Erkkila, E. Martelin, M. Parvinen, M. O. Pentikainen, and L. Dunkel Nuclear Factor-{kappa}B Activation in Human Testicular Apoptosis Am. J. Pathol., January 1, 2002; 160(1): 205 - 218. [Abstract] [Full Text] [PDF]
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T. M. Baetu, H. Kwon, S. Sharma, N. Grandvaux, and J. Hiscott Disruption of NF-{kappa}B Signaling Reveals a Novel Role for NF-{kappa}B in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression J. Immunol., September 15, 2001; 167(6): 3164 - 3173. [Abstract] [Full Text] [PDF]
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F. Chen, V. Castranova, and X. Shi New Insights into the Role of Nuclear Factor-{kappa}B in Cell Growth Regulation Am. J. Pathol., August 1, 2001; 159(2): 387 - 397. [Abstract] [Full Text]
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 E. Ayroldi, G. Migliorati, S. Bruscoli, C. Marchetti, O. Zollo, L. Cannarile, F. D'Adamio, and C. Riccardi Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor {kappa}B Blood, August 1, 2001; 98(3): 743 - 753. [Abstract] [Full Text] [PDF]
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J. Wang, E. G. Brooks, K. B. Bamford, T. L. Denning, J. Pappo, and P. B. Ernst Negative Selection of T Cells by Helicobacter pylori as a Model for Bacterial Strain Selection by Immune Evasion J. Immunol., July 15, 2001; 167(2): 926 - 934. [Abstract] [Full Text] [PDF]
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Y. Zheng, F. Ouaaz, P. Bruzzo, V. Singh, S. Gerondakis, and A. A. Beg NF-{{kappa}}B RelA (p65) Is Essential for TNF-{{alpha}}-Induced Fas Expression but Dispensable for Both TCR-Induced Expression and Activation-Induced Cell Death J. Immunol., April 15, 2001; 166(8): 4949 - 4957. [Abstract] [Full Text] [PDF]
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A.-M. Steff, S. Trop, M. Maira, J. Drouin, and P. Hugo Opposite Ability of Pre-TCR and {{alpha}}{{beta}}TCR to Induce Apoptosis J. Immunol., April 15, 2001; 166(8): 5044 - 5050. [Abstract] [Full Text] [PDF]
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 C. W. Xiao, K. Ash, and B. K. Tsang Nuclear Factor-{{kappa}}B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor {{alpha}}-Induced Apoptosis Endocrinology, February 1, 2001; 142(2): 557 - 563. [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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M. Delgado and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit Expression of Fas Ligand in Activated T Lymphocytes by Regulating c-Myc, NF-{{kappa}}B, NF-AT, and Early Growth Factors 2/3 J. Immunol., January 15, 2001; 166(2): 1028 - 1040. [Abstract] [Full Text] [PDF]
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 M. E. De Paepe, L. P. Rubin, C. Jude, A. M. Lesieur-Brooks, D. R. Mills, and F. I. Luks Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development Am J Physiol Lung Cell Mol Physiol, November 1, 2000; 279(5): L967 - L976. [Abstract] [Full Text] [PDF]
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A. Qadri, C. G. Radu, J. Thatte, P. Cianga, B. T. Ober, R. J. Ober, and E. S. Ward A Role for the Region Encompassing the c'' Strand of a TCR V{alpha} Domain in T Cell Activation Events J. Immunol., July 15, 2000; 165(2): 820 - 829. [Abstract] [Full Text] [PDF]
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L. A. Norian, K. M. Latinis, S. L. Eliason, K. Lyson, C. Yang, T. Ratliff, and G. A. Koretzky The Regulation of CD95 (Fas) Ligand Expression in Primary T Cells: Induction of Promoter Activation in CD95LP-Luc Transgenic Mice J. Immunol., May 1, 2000; 164(9): 4471 - 4480. [Abstract] [Full Text] [PDF]
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K. Matsui, S. Xiao, A. Fine, and S.-T. Ju Role of Activator Protein-1 in TCR-Mediated Regulation of the Murine fasl Promoter J. Immunol., March 15, 2000; 164(6): 3002 - 3008. [Abstract] [Full Text] [PDF]
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F. Kuhnel, L. Zender, Y. Paul, M. K. Tietze, C. Trautwein, M. Manns, and S. Kubicka NFkappa B Mediates Apoptosis through Transcriptional Activation of Fas (CD95) in Adenoviral Hepatitis J. Biol. Chem., February 25, 2000; 275(9): 6421 - 6427. [Abstract] [Full Text] [PDF]
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J. Zhang, B. Ma, A. Marshak-Rothstein, and A. Fine Characterization of a Novel Cis-element That Regulates Fas Ligand Expression in Corneal Endothelial Cells J. Biol. Chem., September 10, 1999; 274(37): 26537 - 26542. [Abstract] [Full Text] [PDF]
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S.-C. Hsu, M. A. Gavrilin, M.-H. Tsai, J. Han, and M.-Z. Lai p38 Mitogen-activated Protein Kinase Is Involved in Fas Ligand Expression J. Biol. Chem., September 3, 1999; 274(36): 25769 - 25776. [Abstract] [Full Text] [PDF]
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J. C. Pratt, M. R. M. van den Brink, V. E. Igras, S. F. Walk, K. S. Ravichandran, and S. J. Burakoff Requirement for Shc in TCR-Mediated Activation of a T Cell Hybridoma J. Immunol., September 1, 1999; 163(5): 2586 - 2591. [Abstract] [Full Text] [PDF]
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I. Rivera-Walsh, M. E. Cvijic, G. Xiao, and S.-C. Sun The NF-kappa B Signaling Pathway Is Not Required for Fas Ligand Gene Induction but Mediates Protection from Activation-induced Cell Death J. Biol. Chem., August 11, 2000; 275(33): 25222 - 25230. [Abstract] [Full Text] [PDF]
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Y. Chen and M.-Z. Lai c-Jun NH2-terminal Kinase Activation Leads to a FADD-dependent but Fas Ligand-independent Cell Death in Jurkat T Cells J. Biol. Chem., March 9, 2001; 276(11): 8350 - 8357. [Abstract] [Full Text] [PDF]
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P. R. Mittelstadt and J. D. Ashwell Inhibition of AP-1 by the Glucocorticoid-inducible Protein GILZ J. Biol. Chem., July 27, 2001; 276(31): 29603 - 29610. [Abstract] [Full Text] [PDF]
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