(Received for publication, October 24, 1995; and in revised form, January 30, 1996)
From the
Engagement of the CD95 (APO-1/Fas) receptor induces apoptosis in
a variety of cell types. However, the nature of the cytotoxic signal
and the intermediate messenger molecules remain to be elucidated. In an
effort to understand CD95-mediated signaling, we assessed possible
changes in the DNA binding activity of NF-B as a result of CD95
engagement in various tumor cells. By performing electrophoresis
mobility shift assays, we show that CD95 can stimulate the DNA binding
activity of NF-
B in a variety of cells, irrespective of their
sensitivity or resistance to CD95-mediated cytotoxicity. Moreover,
deletion of 37 carboxyl-terminal residues from the cytoplasmic domain
of CD95, which abrogates CD95-mediated apoptosis, only marginally
affects NF-
B activation. Taken together, these observations
indicate that CD95 has a function that involves activation of NF-
B
and that appears to be unrelated to its role as an inducer of apoptotic
cell death.
CD95 (APO-1/Fas) is a member of the TNF()/nerve
growth factor receptor superfamily and is currently recognized as the
principal cell surface receptor involved in the transduction of signals
that induce apoptosis in lymphocytes and in a variety of tumor
cells(1) . CD95-mediated apoptosis can be triggered following
engagement of the CD95 receptor by a specific ligand (CD95L) expressed
on activated cytotoxic T cells (1) and by specific anti-CD95
monoclonal antibodies (mAb). Naturally occurring loss of function
mutants of CD95 and CD95 ligand in lpr/lpr and gld mice, respectively(2, 3) , are associated with
lymphoproliferative and autoimmune disease attesting to the importance
of the role that CD95 plays in the maintenance of lymphoid tissue
homeostasis. Although CD95-mediated apoptosis is currently a field of
intense study, little is known about the signaling pathway along which
CD95-mediated cytotoxicity is transduced. The cytoplasmic domain of
CD95 contains a segment known as the ``death domain,''
homologs of which are present within the cytoplasmic domain of the TNF
receptor p55, and several recently isolated intracellular proteins that
interact with CD95(4, 5, 6, 7) .
While expression of the death domain is required for CD95-mediated
cytotoxicity(8) , the full spectrum of its putative functions
remains to be determined, since related sequences have now been
observed in molecules that are not known to be involved in mediating
cell death(9) . Furthermore, despite significant relatedness
between CD95 and TNFRp55, the bulk of evidence currently available
indicates that induction of cell death by these two receptors proceeds
along different pathways(10) .
Expression of CD95 in any given cell type does not invariably indicate that the cell is sensitive to CD95-mediated cytotoxicity. Sensitivity of cells to the death signal induced by CD95 has been shown to be highly variable and to depend in part on their metabolic state(11) . Thus, CD95-mediated cell death appears to be regulated in cell type- and possibly activation/differentiation stage-specific fashion. Interestingly, the function of CD95 does not appear to be exclusively restricted to the induction of apoptosis. One study has shown that engagement of CD95 by mAb can trigger T cell activation(12) , suggesting that CD95 may have functional properties other than cytotoxic signal transduction which remain to be fully explored.
It has been suggested that
signals that induce apoptotic cell death may require activation of
``death genes'' or repression of ``survival
genes''(13, 14) . Because apoptosis mediated by
CD95 occurs within minutes in some cell types(10) , the second
possibility appears more likely. It has been shown, for example, that
glucocorticoid-induced apoptosis of human leukemic cells is caused by
transrepression(14) , and a number of mechanisms have been
proposed to underlie transcription factor-mediated inhibition of gene
expression(15) . In most cases, repression of transcription
involves DNA binding or release by factors which can be visualized by
electrophoretic mobility shift assays (EMSAs). We therefore used EMSAs
to address transcription factor DNA binding activity during
CD95-mediated apoptosis. One potentially attractive candidate
transcription factor is NF-B, because it is inducible and can both
repress and activate transcription of numerous genes whose products are
critical to a variety of biological processes, most notably in
inflammation and immune responses(16) . Using cell lines that
are naturally sensitive or resistant to CD95-dependent apoptosis, we
show that engagement of CD95 can activate the binding of NF-
B to
its specific DNA binding site in a cell type-dependent manner. However,
there appears to be no direct relationship between activation of
NF-
B and the cytotoxic function of CD95. This notion is supported
by the observation that deletion of a portion of the death domain
within the intracytoplasmic region of CD95, which results in abrogation
of CD95-mediated cytotoxicity only minimally affects NF-
B
activation.
Figure 2:
Identification of the classical
NF-B-DNA complex by supershift assays in T24 and Jurkat cells.
Nuclear extracts from T24 and Jurkat cells treated with anti-CD95 mAb
and TNF-
, respectively, were incubated with the probe as indicated
under ``Experimental Procedures'' and further incubated for
45 min at 25 °C with EMSA buffer (lanes 1 and 4)
or with antibody against p65 (lanes 2 and 5) or
against p50 (lanes 3 and 6). The retarded complexes and
nonspecific binding activity are indicated as in Fig. 1B.
Figure 1:
A, sensitivity of T24
cells to CD95-mediated cytotoxicity. T24 cells were incubated for 1 h
in the presence of medium alone or medium supplemented with 10
µg/ml cycloheximide, 25 µM DPI, 1 mM DDC or
with 1 mM DDC and 25 µM DPI (DDC/DPI). Increasing
concentrations of anti-CD95 mAb were then added and cell viability
tested by crystal violet staining 18 h later. Percent cell survival
represents the ratio the of the staining OD value of cells treated with
the different chemicals and anti-CD95 mAb to that of cells treated with
the corresponding chemicals but without anti-CD95 mAb. B,
determination of CD95-induced NF-B DNA binding activity in T24
cells. Nuclear extracts from T24 cells treated with medium alone (lane 2), TNF-
(lane 3), anti-CD95 mAb (lane
4), cycloheximide (lane 5), cycloheximide and anti-CD95
mAb (lane 6), DPI (lane 7), DPI and anti-CD95 mAb (lane 8), and DPI, DDC, and anti-CD95 mAb (lane 9) as
described under ``Experimental Procedures'' were subjected to
EMSAs using radiolabeled NF-
B consensus binding site
double-stranded oligonucleotide. The retarded complexes obtained are
numbered 1-5. The open arrow indicates
nonspecific binding to the probe.
, medium;
,
cycloheximide;
, DPI;
, DDC;
,
DDC/DPI.
Although T24 cells are resistant
to cytotoxic signals transduced by CD95 and TNF receptors ( Fig. 1and data not shown), engagement of both receptors in these
cells results in the activation of NF-B. To determine whether
sensitization of T24 cells to CD95 might alter NF-
B binding
activity, electrophoretic mobility shift assays were performed using
lysates of T24 cells that had been triggered with anti-CD95 antibody
following exposure to CHX or DPI. Both CHX and DPI pretreatment
augmented T24 cell sensitivity to CD95-mediated cytotoxicity (Fig. 1A). Pretreatment of the T24 cells with
cycloheximide increased the effect of anti-CD95 mAb on NF-
B DNA
binding activity (Fig. 1B). However, this is probably
due to the additive effect of cycloheximide and CD95 engagement, since
cycloheximide alone can activate NF-
B (Fig. 1B and (20) ). DPI pretreatment of the T24 cells, on the other hand,
inhibits the effect of anti-CD95 mAb on NF-
B binding activity (Fig. 1B). The low degree of NF-
B activation in
this case is most likely due to the effect of DPI treatment alone (Fig. 1B).
We have shown previously that exposure of
cells to diethyldithiocarbamate (DDC) induces resistance to
CD95-mediated death by increasing intracellular
O concentration as a result of the
inhibition of CuZn-superoxide dismutase(18) . Preincubation of
T24 cells with DPI and DDC has been observed to result in the
restoration of resistance to CD95 from DPI-induced sensitivity (Fig. 1A and (18) ). To determine whether this
reversal of phenotype correlates with any detectable modification in
NF-
B binding, lysates of T24 cells triggered with anti-CD95 mAb
following pretreatment with DPI and DDC were subjected to EMSAs. The
NF-
B DNA binding activity was not observed to be increased, in
contrast to anti-CD95 mAb treatment alone. The complexes obtained were
comparable with the ones seen following treatment of cells with DPI
alone or with DPI and anti-CD95 mAb (Fig. 1B).
The
above observations indicate that engagement of CD95 by mAb in T24 cells
can activate NF-B. Importantly, however, there appears to be no
correlation between the NF-
B activation and cell sensitivity or
resistance to CD95. Although engagement of CD95 in untreated, resistant
cells activates NF-
B, activation is inhibited by pretreatment with
a combination of DPI and DDC, which does not alter cell resistance to
CD95-mediated killing. NF-
B activation in T24 cells that are
sensitized to CD95, on the other hand, is not related to the
sensitivity of the cells but rather depends on the reagent used to
induce sensitivity. Thus, whereas both CHX and DPI sensitize T24 cells
to CD95-mediated cytotoxicity, CHX augments while DPI inhibits
CD95-induced NF-
B activation.
To address the relationship
between engagement of CD95 and NF-B activation in different cell
types, we performed DNA mobility gel shift analysis using two cell
lines that are naturally sensitive to CD95, the T cell leukemia Jurkat,
which constitutively expresses CD95 and TNFR, and the human melanoma MC
stably transfected with the CD40-Fas fusion protein(10) . The
CD40-Fas fusion protein consists of the extracellular domain of CD40
and the intracellular and transmembrane domains of CD95. CD95-mediated
cell death can be conveniently induced by soluble recombinant CD8-CD40L
(CD8-gp39) fusion protein overproduced in COS cells(10) . When
Jurkat cells were treated with anti-CD95 mAb, no activation of
NF-
B DNA binding was observed, in contrast to TNF-
treatment (Fig. 3). Cycloheximide treatment alone was observed to activate
NF-
B (mainly complex 3) in Jurkat cells, but no increase in the
binding activity was seen following incubation of cycloheximide-treated
cells with anti-CD95 mAb (Fig. 3). The effect of cycloheximide
appears to be nonspecific, possibly resulting from the inhibition of
I-
B, the natural repressor of NF-
B(21) . Contrary to
Jurkat cells, MC40-Fas transfectants display activation of NF-
B
binding activity following stimulation with soluble CD40L (Fig. 4).
Figure 3:
Determination of CD95-induced NF-B
DNA binding activity in Jurkat cells. Nuclear extracts from Jurkat
cells treated with medium alone (lane 2), TNF-
(lane
3), anti-CD95 mAb (lane 4), cycloheximide (lane
5), cycloheximide and anti-CD95 mAb (lane 6), as
described under ``Experimental Procedures,'' were subjected
to EMSAs using radiolabeled NF-
B consensus binding site
double-stranded oligonucleotide. The retarded complexes and nonspecific
binding activity are indicated as in Fig. 1B.
Figure 4:
Determination of CD95-induced NF-B
DNA binding activity in MC-CD40-Fas cells. Nuclear extracts from
MC-CD40-Fas treated with medium alone (lane 2) or with
CD8-CD40L (CD8-gp39) fusion protein (lane 3) generated in COS
cells as described under ``Experimental Procedures'' were
subjected to EMSAs using radiolabeled NF-kB consensus binding site
double-stranded oligonucleotide. The retarded complexes obtained and
nonspecific binding to the probe are indicated as in Fig. 1B.
These observations support the notion that
triggering of CD95 augments the DNA binding activity of NF-B in
some but not all cell types and that there is no correlation between
activation of binding and cell sensitivity to CD95-mediated killing.
Moreover these results may explain the recent report by Schulze-Osthoff et al.(22) that triggering CD95 overexpressed in the
CD95 and TNF-sensitive cell line L929 does not activate NF-
B,
whereas engagement of the TNFR by TNF-
does.
Figure 5:
Induction of cell death and NF-B DNA
binding activity in MC-CD40-Fas and MC-CD40-Fas deletion mutant
transfectants. A, wild type MC-CD40-Fas and deletion mutants
are represented on the left panel. Sequences encoding the
extracellular domain of CD40 were fused to sequences encoding wild type
transmembrane (TM, amino acids 152-174) and
intracytoplasmic (amino acids 175-319) domains of CD95. The
mutants generated by polymerase chain reaction were truncated at amino
acids 181, 215, 247, 282, and 311 and designated R1, R2, R3, R4, and
R7, respectively. The death domain (amino acids 215-304) is
indicated by shading. Induction of apoptotic cell death was
performed by incubating MC transfectants for 18 h with COS cell
CD8-gp39 supernatant, and the viability was determined using crytal
violet staining. + indicates >80% cell death, whereas -
indicates <20% cell death during the incubation time period. B, determination of CD95-induced NF-
B DNA binding
activity in MC-CD40-Fas deletion mutant transfectants. Nuclear extracts
from deletion mutants MC-CD40-FasR1 (lane 5), MC-CD40-FasR2 (lane 6), MC-CD40-FasR3 (lane 7), MC-CD40-FasR4 (lane 8), and MC-CD40-FasR7 (lane 9) treated with
CD8-CD40L (CD8-gp39) fusion protein were subjected to EMSAs using
radiolabeled NF-
B consensus binding site double-stranded
oligonucleotide. Nuclear extracts from MC-Neo and MC-CD40-Fas cells
treated with CD8-gp39 provided a negative and positive control
respectively, and extracts from MC-CD40-Fas cells incubated in medium
alone provided a second negative control. The retarded complexes and
nonspecific binding to the probe are indicated as in Fig. 1B.
The functional
significance of NF-B activation by CD95 is presently unknown.
However, the present observations support the notion that functions
other than triggering of apoptosis are mediated by CD95, which may be
relevant to studies on the interaction of CD95 with intracellular
proteins. Our results demonstrate the presence of several complexes by
EMSAs following CD95 triggering that probably consist of different
NF-
B/Rel related factors present in various homo- or heterodimeric
combinations. The putative effects of these factors and the identity of
their target genes can presently be only speculative. It is possible,
for example, that some of the observed complexes may selectively
activate or repress gene transcription or alternatively have both
stimulatory and inhibitory effects on different genes in the same
cell(23) . Characterization of these factors and their target
genes may help to uncover hitherto unrecognized functions of CD95.