From the Department of Biochemistry, Trinity College, Dublin 2, Ireland
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ABSTRACT |
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The role of ceramide as a second messenger in
tumor necrosis factor (TNF)-mediated signal transduction has been much
debated. It is supported by recent reports describing an expanding
number of potential targets for this lipid, but is opposed by those
describing how ceramide is not necessary for many TNF-mediated cellular
events. In this paper, we directly compare the effects of the
cell-permeable ceramide analogue, N-acetylsphingosine
(C2-ceramide), with TNF, on NFB function, a
transcription factor whose activation is central to many TNF-mediated
effects. We describe how C2-ceramide failed to drive
B-linked chloramphenicol acetyltransferase gene expression in either
HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it had no
effect on TNF-mediated transcription of this reporter gene. However,
electrophoretic mobility shift analysis following cell stimulation with
this ceramide analogue revealed a dose-responsive activation of NF
B,
which was not apparent following cell treatment with the inactive
dihydro form. Activated complexes from treated cells were shown to
contain predominantly the p50 subunit, in contrast to complexes from
TNF-treated cells, where both p50 and p65/RelA subunits were present.
The specific activation of p50 homodimeric complexes by
C2-ceramide, which are known to lack trans-activating
activity, was strongly suggested from these data. Further
investigations revealed that C2-ceramide had only a
marginal effect on I
B
degradation but strongly promoted the
processing of p105 to its p50 product as revealed by immunoblot
analysis. The increase in p50 arising from the processing of its p105
precursor was further established from p105/p50 ratios obtained by
scanning densitometric analysis of bands from immunoblots. TNF, on the other hand, stimulated both I
B
degradation and p105 processing, in accordance with previous findings. Furthermore, the effect of TNF on
NF
B activation was rapid, whereas C2-ceramide required an optimal treatment time of 1 h. Interestingly, TNF was found to
increase ceramide in cells but only after a 1-h contact time. Our data
therefore suggest that ceramide promotes the activation of NF
B
complexes that lack transactivating activity by enhanced processing of
p105.
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INTRODUCTION |
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Tumor necrosis factor
(TNF)1 is a pleiotropic
cytokine that induces a variety of cell type-specific events including
proliferation, differentiation, necrosis, and apoptosis (1). It is
among a range of agents that have been reported to increase levels of the neutral lipid, ceramide, in cells (2-12). This has led to the
proposal that ceramide may be a second messenger for TNF, where
cell-permeable analogues of ceramide can mimic several TNF effects,
including activation of the transcription factor, NFB (13-15).
Ceramide has also been shown to activate several protein kinases
(16-23), a protein phosphatase (24), and the nucleotide exchange
protein, Vav (25), and to induce apoptosis (26-30).
NFB is a member of the Rel family of transcription factors (31). It
is activated by a wide range of stimuli, including TNF, interleukin-1,
UV irradiation (reviewed in Ref. 32), and chemotherapeutic drugs (12,
33), all of which have been shown to increase ceramide levels in cells
(30). The mechanism of NF
B activation has been the subject of much
scrutiny. It binds to a discrete nucleotide sequence (5'-GGGACTTCC-3')
in the upstream regions of genes that code for mediators of the immune,
acute phase, and inflammatory responses, thereby regulating their
expression (32). The activation of NF
B can occur by two mechanisms.
The prototypical pathway involves its liberation from an inactive complex with the inhibitor protein, I
B, which resides in the cytosol
(31, 32). Several forms of I
B have been identified (34), with
particular attention focusing on I
B
. In resting cells, it is
complexed to the NF
B heterodimer, typically comprising p50 and
p65/RelA subunits. Following cell stimulation, a pathway is activated
that culminates in the phosphorylation of I
B
on two serine
residues (Ser32 and Ser36) (35), which tags it
for degradation by the proteosome (36), leading to the nuclear
translocation of the NF
B heterodimer. Recently, a pathway activated
by TNF involving TRAF-2, an NF
B-inducing kinase, and two novel
kinases termed IKK-1 and IKK-2 (or IKK-
and IKK-
) has been
described (37-40), with both IKKs phosphorylating I
B
(39, 40).
Another less well defined pathway for NF
B activation has also been
described (31). The p50 subunit of NF
B is generated following
processing from its precursor, p105 (41). Proteolysis results in the
removal of an inhibitory C-terminal domain containing seven ankyrin
repeats, a motif present in I
B proteins, revealing a nuclear
translocation signal for this subunit. Specifically, a 68-amino acid
sequence in the C-terminal PEST domain of p105 contains multiple
serines that are phosphorylated (42) by an as yet unidentified kinase,
prior to proteosome-directed proteolytic degradation (43). Processing
of p105 complexed to p65/RelA would allow the heterodimer to
translocate into the nucleus in an analogous manner to that which
occurs following I
B
proteolysis. In addition, p50 homodimers,
which have been shown to be transcriptional repressors (44), would also
translocate to the nucleus. Once in the nucleus, p50 could complex with
p65/RelA and c-Rel, giving rise to heterodimers with transcriptional
potential. p105 processing has been shown to be stimulated by TNF,
phorbol esters, and double-stranded RNA (45, 46). The activation of
NF
B family members may therefore be regulated by this pathway in
parallel with that mediating I
B degradation in response to the same
signal.
The mechanism by which ceramide can trigger either of the pathways
leading to NFB activation is unresolved, with conflicting results
being presented (47-53). Here, we have compared the effect of a
commonly used short chain cell-permeable analogue of ceramide, N-acetylsphingosine (C2-ceramide), with TNF in
four independent assays of NF
B function. We have found that although
C2-ceramide can activate NF
B as judged by gel shift
analysis, prolonged treatment times are required and only a marginal
increase in I
B
degradation is observed. Furthermore, there is no
effect on NF
B-linked reporter gene expression. However,
C2-ceramide can induce p105 processing, with NF
B
complexes predominantly comprising p50 homodimers. In contrast,
TNF-activated complexes contain both p50 homodimeric and p50/p65
heterodimeric forms of NF
B. TNF promotes both I
B
and p105
processing and as expected, induces NF
B-linked gene expression. Our
results may therefore help to resolve some of the controversy in this
area, in that although ceramide does not appear to be involved in the
pathway leading to I
B
phosphorylation and degradation, it may
signal p105 processing in response to TNF.
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EXPERIMENTAL PROCEDURES |
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Materials--
HL60 and Jurkat T cells (both obtained from the
European Collection of Animal Cell Culture, Salisbury, UK) were grown
in suspension culture in RPMI 1640 supplemented with 10% fetal calf
serum, penicillin/streptomycin (100 units/ml and 100 mg/ml,
respectively), and L-glutamate (final concentration, 2 mM), all obtained from Life Technologies, Inc. (Paisley,
UK). Recombinant human TNF was a gift from Zeneca Pharmaceuticals Ltd. (Macclesfield, UK). Poly(dI-dC) was from Pharmacia Biosystems (Milton Keynes, UK), T4 polynucleotide kinase and oligonucleotide containing the consensus sequence (5'-GG GAC TTT CC-3'), corresponding to the
light chain enhancer motif, were purchased from Promega (Southampton, UK). [
-32P]ATP (3000 Ci/mmol),
([14C]chloramphenicol (56 mCi/mmol), and ECL reagent were
from Amersham Pharmacia Biotech (Aylesbury, UK). Diacylglycerol kinase
was from Calbiochem (UK). Rabbit polyclonal antibody preparations to
the NF
B subunits, RelA/p65, and p50, were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA) and Dr. Jean Imbert (INSERM,
Marseille, France) (where indicated). Polyclonal antibodies (41) raised against an N-terminal peptide (residues 2-15) of the p105 protein (which also recognized its proteolysis product, p50, containing this
N-terminal sequence) were generously supplied by Dr. Alain Israel
(Institut Pasteur, Paris Cedex 15, France), and monoclonal antibodies
to the inhibitor protein, I
B-
, were from Dr. Ron Hay (St.
Andrews, Scotland). The pCATTM-Promoter plasmid was a gift from Dr.
Tim Bird (Immunex Corporation, Seattle, WA). Mutant NF
B
oligonucleotide was from Santa Cruz Biotechnology Inc.
C2-dihydroceramide (N-acetyldihydrosphingosine)
was from Matreya Inc. (Pleasant Gap, PA). All other reagents were
purchased from Sigma (Poole, Dorset, UK).
Cell Culture-- For treatments, cells in late log phase of growth were resuspended in serum-free medium at a concentration of 5 × 106/ml (2.5 × 106/ml for whole cell extracts) and incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air. Following stimulation (60 min unless otherwise stated), incubations were discontinued by the addition of ice-cold phosphate-buffered saline, and either nuclear or whole cell extracts were prepared as described previously (33). Protein determinations were made using the Bradford assay with bovine albumin as standard.
Transfection Studies--
The transactivating potential of
activated NFB complexes was assessed following transfection of cells
with a plasmid containing five NF
B consensus sequences upstream of a
chloramphenicol acetyltransferase reporter gene. Jurkat T cells were
transfected as described previously (33). HL60 cells were transfected
by electroporation (54) with modifications; cells were washed with 5 units/ml heparin post-transfection followed by incubation in medium
supplemented with 20% serum for 24 h. Following treatment
(indicated in figure legends), extracts prepared from harvested cells
(1 × 106) were assayed for CAT activity (33).
Statistical significance was evaluated by employing Student's
t test for unpaired data.
Electrophoretic Mobility Shift Assays--
Nuclear NFB was
assessed by the electrophoretic mobility shift assay using a 22-base
pair oligonucleotide containing the human
light chain enhancer
motif, which had previously been end-labeled with
[
-32P]ATP as described (33). Typically, 2-4 mg of
nuclear extract protein was incubated with radiolabeled oligonucleotide
(10,000 cpm) at room temperature for 30 min using conditions as
described previously (33). NF
B complexes were resolved on 5%
acrylamide gels and identified following autoradiography. To identify
the subunit components of activated NF
B complexes and the
specificity of the binding reaction, supershift analysis and
competition studies were carried out as described previously (33),
using antibodies described under "Experimental Procedures" to
individual NF
B subunit components and mutant/wild type unlabeled
NF
B consensus sequence, respectively.
Western Blot Analysis--
Equal amounts of whole cell lysate
protein (as indicated) were resolved by SDS-polyacrylamide gel
electrophoresis and transferred onto nitrocellulose, and IB
or
p50/p105 immunoblot analysis was performed as described previously
(33). Secondary antibody was used at a dilution of 1:1000 and 1:2000
for I
B
and p50/p105, respectively. The blots were developed by
ECL according to the manufacturer's recommendations.
Lipid Studies--
Ceramide was quantified by the diacylglycerol
kinase assay (10) with modifications as described (33). The level of
ceramide in HL60 cells following treatment with TNF or not (control)
was determined by comparison with a standard curve generated with known
amounts of ceramide (ceramide type III; Sigma).
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RESULTS |
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Neither C2-ceramide nor Its Dihydro Form Stimulate
B-driven Gene Expression--
In our first experiments with
C2-ceramide, we attempted to mimic the stimulatory effect
of TNF on an NF
B-linked reporter gene construct. HL60 or Jurkat were
therefore transfected with a gene construct containing five NF
B
sites upstream of a CAT reporter gene. Transfected cells were then
stimulated with C2-ceramide or TNF. At 10 µM
C2-ceramide no increase in CAT activity was observed over
control values (unstimulated cells) in either Jurkat (Fig. 1A) or HL60 cells (Fig.
1B). 30 ng/ml TNF, as expected, stimulated NF
B-driven
gene expression, causing a 4-6-fold increase over controls in Jurkat T
cells. A 2-fold stimulation over control levels was seen in HL60 cells.
Co-incubation of Jurkat with both C2-ceramide and TNF
resulted in a similar response to that seen with TNF alone (Fig.
1C). These results suggested that treatment of cells with
ceramide had no effect on NF
B-linked gene expression.
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C2-ceramide, but Not Its Dihydro Congener, Activates
NFB in Both HL60 and Jurkat T Cells--
We next tested whether
ceramide could activate NF
B in these cells, as has been reported by
others (13-15). C2-ceramide dose-dependently activated NF
B in HL60 cells, as was demonstrated by the detection of
protein-DNA complexes in nuclear extracts from ceramide-treated cells
(Fig. 2A). This effect was
evident from 1 µM and contact times of 1 h were
required to see an effect. Stimulation of HL60 cells with both
C2-ceramide and TNF resulted in a modest potentiation of
NF
B activation in comparison with that seen with TNF alone (Fig.
2A, lanes 7 and 8). The dihydro form
of C2-ceramide (which should be ineffective, because it
lacks a critical 4,5-trans double in the sphingoid base
backbone) was inactive (Fig. 2B). TNF strongly activated
NF
B in both cell types (Fig. 2, A and C,
lane 7) and was found to increase ceramide levels in HL60
causing a 2.64 ± 0.29-fold increase over control values after
1 h of stimulation, as shown in Fig. 1C.
C2-ceramide also activated NF
B in Jurkat T cells over a
similar concentration range effective in HL60 cells and a contact time
of 1 h (Fig. 2D).
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NFB Complexes Activated by C2-ceramide Contain
Predominantly p50 Subunit--
We next examined the NF
B complex
activated by C2-ceramide in more detail. Binding
specificity of activated complexes from C2-ceramide-treated
HL60 cells was demonstrated by competition studies. Excess unlabeled
oligonucleotide containing the NF
B consensus sequence inhibited the
appearance of retarded complexes, whereas a mutant oligonucleotide had
no effect at equivalent concentrations (Fig.
3A). We then determined the
composition of the activated complexes from HL60 cells treated with
either C2-ceramide or TNF. To achieve this supershift
analysis was performed using specific antisera to p50 and p65/RelA. The
complexes were electrophoresed further than usual to optimize
resolution. Fig. 3B shows that the complexes activated by
TNF differ markedly from those induced by C2-ceramide
(compare lanes 4 and 1). TNF induces two main
complexes. Treatment of extracts with anti-p65 antiserum inhibited the
formation of the upper complex (lane 5), whereas anti-p50
antiserum affected both upper and lower complexes (lane 6)
and caused a further retardation of the DNA probe. In contrast, the
C2-ceramide complex was only marginally affected by the
anti-p65 antiserum (lane 2). However, this complex was
almost completely supershifted by anti-p50 anti-serum (lane
3), indicating that p50 was much more prevalent than p65/RelA in
the C2-ceramide-activated NF
B complex, whereas both were
present in the TNF-activated complex.
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C2-ceramide Has a Marginal Effect on IB
Degradation but Promotes p105 Processing in HL60 Cells--
HL60 cells
treated with C2-ceramide or TNF at concentrations that
resulted in NF
B activation were next examined for degradation of
I
B
. A rapid and marked degradation was observed following stimulation with TNF (Fig. 4A)
as determined by immunoblotting. The presence of a doublet (lane
2), prior to complete degradation, was most likely because of the
phosphorylation of this protein. I
B
appeared to be completely
degraded by 15 min (lane 3). Resynthesis restored I
B
levels within 60 min to the level of the unstimulated cells. In
contrast, C2-ceramide treatment resulted in a more marginal degradation of I
B
, complete degradation not being evident at any
point (lanes 6-8).
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DISCUSSION |
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Ceramide has emerged as a second messenger implicated in multiple
cellular responses, such as apoptosis, cell cycle arrest, and
differentiation (reviewed in Ref. 30). Although its role is an issue of
some debate, ceramide may function as a selective mediator of the cell
killing effects of TNF, in addition to other cellular events in
response to this cytokine. Because of the controversy that surrounds
the role of this neutral lipid in TNF-mediated signaling, we decided to
compare the signaling events between TNF and ceramide that might
culminate in the activation of the transcription factor, NFB. In our
study, we investigated the ability of C2-ceramide to
stimulate
B-linked gene expression. C2-ceramide has
previously been reported to potentiate and mimic TNF action in this
response (13-15). We were unable to stimulate
B-linked CAT activity
with this compound, however. This was in agreement with an earlier
report where 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (a
closely related analogue) failed to induce transactivation in a
reporter gene assay in Jurkat T cells either alone or in combination
with TNF (50). The absence of CAT stimulation can now be explained by
our observation that selective activation of p50 occurs in response to
ceramide. Specifically, C2-ceramide and not its inactive
congener caused a dose-responsive activation of NF
B in the
hematopoetic cell line, HL60. On closer examination it was revealed
that the pattern of Rel subunits in ceramide-activated complexes was
distinct from that seen in TNF-activated complexes, with p50
predominating. The p50 antiserum employed was partially selective for
p50 homodimeric forms of
NF
B,2 and the data
obtained indicated that this subunit might be selectively activated in
response to ceramide. We addressed p50 generation from the proteolytic
processing of its precursor form, p105, as a possible target of
ceramide, using an antibody that recognized both the parent protein and
its cleaved product, the p50 subunit, and furthermore, compared our
data with that obtained with TNF. We found a marked preference for p105
processing over I
B
degradation by ceramide, in contrast to TNF,
which although capable of increasing p105 processing also caused a
rapid degradation of I
B
(preceded by an apparent
phosphorylation), in line with the classical pathway of NF
B
activation. Our data therefore indicated that although ceramide
activates NF
B, the subunit composition differs from that observed
with TNF.
A number of other studies have examined whether ceramide can activate
NFB and probed the possible mechanism involved. Ceramide analogues
have been found to activate NF
B (13-15), to potentiate the
activation in response to TNF (47), or to have no effect (29, 47, 50,
51). The reasons for such marked discrepancies are unclear. Two studies
that did not observe any effect were carried out in Jurkat (29, 50),
which we found to be less responsive than HL60 cells. We also found
that relatively high concentrations were needed to observe an effect
and that treatment times of 1 h were required. These conditions
may not be possible in some cell lines, where ceramide may induce
apoptosis. Under the conditions used in our studies, no apoptosis
was observed (not shown). Most recently, Gamard et al. (29)
demonstrated that C2-ceramide did not induce I
B
degradation in Jurkat T cells. This study also found no effect of this
compound on NF
B activation, as assessed by gel shift analysis,
although the cells were exposed for only 20 min at a
C2-ceramide concentration of 40 µM. We found that 40 µM C2-ceramide activated NF
B in
Jurkat T cells but that a 1-h contact time was required. A similar
result was obtained in ML-1a leukemia cells (51), where a minimum
contact time of 60 min was required to observe NF
B activation. The
observation that a 1-h contact time was required may be significant,
because TNF was found to increase ceramide levels in cells after a 1-h stimulation. As reported (29, 51), NF
B activation by TNF occurs much
more rapidly than this, indicating that immediate activation of NF
B
by TNF is unlikely to involve ceramide. It is possible that p105
processing by TNF, which we found to occur at a later time compared
with I
B
degradation, may be mediated by ceramide. Different rates
of I
B
and p105 processing in response to TNF stimulation have
also been observed by Melitts et al. (45). p105 processing
as a mechanism of NF
B activation has received much less attention
than the pathway involving I
B
degradation. Phosphorylation of
p105 is required for its processing (42), similar to I
B-
.
However, unlike I
B
, a novel E3 component of the proteosome is
involved in p105 proteolysis (55). Phosphorylation occurs on multiple
serines in the C-terminal PEST domain (42). The kinase or phosphatase
(which would be inhibited) responsible for this modification is not
known. Because ceramide has been shown to activate an as yet
unidentified ceramide-activated protein kinase (16) as well as protein
kinase C
(18), either may be involved. Our results would argue that
the component of NF
B activation by TNF, which involves p105
processing rather than I
B
degradation, could be mediated by
ceramide.
The enzyme(s) responsible for ceramide generation by TNF is still
unresolved. Both acidic and neutral sphingomyelinase activities have
been detected in extracts from TNF-treated cells (56). Evidence
suggesting a role for acidic sphingomyelinase in NFB activation has
been presented (56). This has been disputed in two studies, however,
demonstrating that TNF is able to activate NF
B in fibroblasts from
Niemann Pick patients that lack acidic sphingomyelinase and in acid
sphingomyelinase-deficient knockout mice (48, 52). In addition, another
study has shown that an inhibitor of acidic sphingomyelinase, SR33557,
did not inhibit NF
B activation by TNF (51). We found no effect of
TNF on either neutral or acidic sphingomyelinase activities in HL60
cells (data not shown). An alternative mechanism must therefore be
occurring that gives rise to the detected increase in ceramide, which
could involve inhibition of ceramide metabolism or its increased
biosynthesis (30).
The ability of ceramide to increase p50 homodimers in the relative
absence of p50/p65 heterodimers may have consequences for gene
expression. p65 is a potent transcription activation subunit, whereas
p50 is not, because of the absence of a trans-activator domain (31,
32). In fact, evidence exists where p50 homodimers might function as
negative regulators of B-dependent transcription in vivo (44). Thus the ratio of transcriptionally active to inactive dimeric complexes is paramount in determining gene expression, in addition to the relative affinity of distinct NF
B binding sites
for these transcription factors (57). Furthermore, p105 processing may
also contribute indirectly to
B binding by generating dimers that
associate with newly synthesized I
B, as has been previously
suggested (42, 58). We found no inhibitory effect on the expression of
the NF
B-linked reporter gene, indicating that the ability of TNF to
rapidly increase p50/p65 heterodimers would overcome any inhibitory
effect of p50 homodimers. Much higher levels of p50 homodimers may be
required to see inhibition, as has been shown in studies on the
interleukin-2 promoter in T cells (44).
The ability of C2-ceramide to increase p50 homodimers may,
however, account for some of the anti-proliferative properties reported
for ceramide and analogues, such as down-regulation of c-myc
gene transcription (27). In support of this proposal, a decrease in the
rate of c-myc gene transcription has been directly related
to a significant increase in the binding of p50 homodimers, which fail
to transactivate the c-myc promoter (59). Down-regulation of
c-myc expression has previously been shown to reflect
perturbations in regulatory processes contributing to growth arrest and
apoptosis (reviewed in Ref. 60), and ceramide has been shown to induce down-regulation of c-myc mRNA levels (by an unknown
mechanism), acting possibly via ceramide-activated protein phosphatase
(27). C2-ceramide has also been shown to suppress the
expression of the cytochrome P-450 2C11 (CYP2C11) gene (61). The
protein product of this gene is a member of a family of enzymes whose
content has been found to decrease in hepatic cells during infection
and inflammation. Ceramide is thought to mediate this down-regulation, although a mechanism is not suggested. Because the gene for CYP2C11 is
NFB regulated, we would hypothesize that its down-regulation may
involve increases in p50 homodimers.
The pro-apoptotic effects of ceramide could also involve the generation
of p50 homodimers. NFB activation has been shown to be
anti-apoptotic (62), presumably through the induction of anti-apoptotic
genes. An increase in p50 homodimers by ceramide could block the
expression of such genes and thereby promote apoptosis.
In conclusion, our study indicates that the activation of NFB by
ceramide involves p105 processing in preference to I
B
degradation. This phenomenon may be involved in that aspect of NF
B
activation by TNF that comprises the generation of p50 homodimers rather than I
B
degradation followed by release of the p50/p65 heterodimer.
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ACKNOWLEDGEMENTS |
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Anti-sera to the NFB p50 subunit component
and its p105 precursor (peptide 1141) were generously provided by Dr.
Jean Imbert (INSERM, Marseille, France) and Dr. Alain Israel (Institut
Pasteur, Paris, France), respectively. A monoclonal antibody to the
I
B inhibitor protein was a gift from Prof. Ron Hay (University of St. Andrews, Scotland). We are grateful to the Cancer Research Advancement Board and Zeneca Pharmaceuticals for financial support and
acknowledge Dr. Steve Foster's invaluable contribution to this
work.
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FOOTNOTES |
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* This work was supported by a grant from the Cancer Research Advancement Board, Ireland (to M. P. B. and L. A. J. O.) and by funding from the Cardiovascular, Metabolism and Musculoskeletal Research Department, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.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. Tel.: 353-1-608-2449;
Fax: 353-1-677-2400; E-mail: mpboland{at}tcd.ie.
1
The abbreviations used are: TNF, tumor necrosis
factor; NFB, nuclear factor
B; C2-ceramide,
N-acetylsphingosine; CAT, chloramphenicol acetyltransferase.
2 J. Imbert, personal communication.
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REFERENCES |
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