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RI-Mediated Induction of TNF-
Gene Expression in the RBL-2H3 Mast Cell Line: Regulation by a Novel NF-
B-Like Nuclear Binding Complex1
*
Unité Immuno-Allergie, Institut Pasteur,
Unité 363, Institut National de la Santé et de la Recherche Médicale, Institut Cochin de Genetique Moleculaire, Hôpital Cochin, Paris, France; and
Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) regulates TNF-
gene expression. Antigenic stimulation of
RBL-2H3 cells led to an increase in newly synthesized TNF- mRNA that
was dependent on continuous receptor aggregation and did not require de
novo protein synthesis. Kinetic analysis showed that maximal levels
were achieved at 60 min and waned by 180 min of stimulation.
Concomitant with the transcriptional activation of the TNF- gene,
the rapid appearance and disappearance of a previously uncharacterized
nuclear NF-
B DNA binding activity, comprised of two distinct protein
complexes, were observed. These protein complexes bound to NF-B
sites within the TNF- gene and contained novel proteins (three
species of Mr between 90,000–110,000)
distinct from the classical proteins in NF-B complexes. The induced
NF-B binding activity required continuous receptor stimulation and
induced NF-B-dependent reporter gene expression. Consistent with a
role for the novel NF-B nuclear binding activity in TNF- gene
expression, deletion of several 5' B elements in the TNF-
promoter abolished all measurable FcRI-dependent induction of a
reporter construct. Pharmacologic agents that inhibited the NF-B
binding activity also inhibited TNF- mRNA expression. Our results
demonstrate that a novel NF-B-like nuclear binding activity plays an
important role in regulation of the rapid and transient transcriptional
activation of the TNF- gene via FcRI. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI3 on mast cells and
basophils induces the synthesis and secretion of an array of cytokines
with proinflammatory and immunoregulatory properties (1). One of the
cytokines produced is TNF-, an inflammatory mediator with multiple
biologic functions (2). Mast cell-derived TNF- has a protective role
in acute septic peritonitis and is involved in the recruitment of
neutrophils for clearance at sites of bacterial infection (3, 4). It is
also clear that the production of TNF- has a detrimental role in
allergy and asthma as a mediator of late phase inflammatory reactions
(5). In mast cells, TNF- has been found prestored in cytoplasmic
granules and is released with histamine and other preformed mediators
within minutes following antigenic stimulation, suggesting a potential
role in early inflammation as well (6). In addition, there is a
prolonged secretion of TNF-, since it is newly synthesized and
secreted via a regulated Golgi secretory pathway that requires
continuous aggregation of receptors (7).
Due to its potent and pleiotropic actions as an inflammatory mediator
and the delicate balance of beneficial and toxic effects, the
production of TNF- is tightly regulated in many cell types,
including mast cells (8, 9, 10). Regulation involves both transcriptional
and post-transcriptional mechanisms (10, 11, 12). Transcriptional
activation of the TNF- gene expression is induced by diverse
stimuli, some relatively nonspecific such as LPS and PMA, and others
more specific such as TNF-, IFN-
, and Ag activation of FcR and
TCR (10). Multiple consensus sequences for transcription factors have
been identified within the TNF- gene loci (9, 13, 14, 15). The
transcription factors implicated are differentially regulated and
appear to depend, in addition to the stimulus, on the cell type and the
species studied. For example, both murine and human promoters contain
several binding sites for the transcription factor NF-B. In murine
macrophages, LPS induction of TNF- gene expression was mediated by
the binding of NF-B to its target DNA sequence (16, 17), while, in
contrast, analysis of the human promoter sequence suggested that
NF-B consensus binding sequences are neither required nor sufficient
for LPS- or virus-induced TNF- gene expression (18). Regulation in
murine macrophages and rat astrocytes also seems to involve a B
element downstream of the TNF- polyadenylation site (13, 15).
Recently, a NF-AT binding site in the human TNF- promoter was shown
to confer a stimulation-dependent induction of this gene in lymphocytes
and mast cells (19, 20). In T lymphocytes the NF-AT binding element was
found to function coordinately with an upstream localized
cAMP-responsive element that bound activating transcription factor 2
and Jun family proteins (21, 22). In addition to NF-B and NF-AT
other transcription factors, such as AP-1 (23), ets (24),
and C/EBPß (25), also appear to be involved in TNF- gene
expression.
In this study we focused on the role of NF-B binding elements in
activating transcription of the TNF- gene in RBL-2H3 cells
stimulated via FcRI. We provide evidence for a mechanism implicating
the transient activation of the DNA binding activity of a previously
uncharacterized NF-B-related binding complex in the nucleus as
important to transient activation of the TNF- gene in response to
aggregation of FcRI.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Supernatants from serum-free hollow-fiber cultures of the
hybridoma Hi-DNP--26.82 (26) were used as a source of
anti-DNP-specific IgE at a dilution of 1/200 for all sensitization
experiments. Rabbit serum to TNF- was purchased from Genzyme
(Cambridge, MA). Abs to NF-B complex proteins, p50 (N and C
terminus), p65 (Rel-A, N and C terminus), Rel-B, Rel-C, and p52 and a
pan-specific Ab to NF-AT complex proteins were purchased as supershift
reagents from Santa Cruz Biotechnologies (Santa Cruz, CA). Additional
Abs to the p50 and p65 constituent of NF-B were provided by Drs. A.
Israel (Institut Pasteur, Paris, France) and U. Siebenlist (National
Institutes of Health, Bethesda, MD). DNP43-BSA was a gift
from Dr. A. Prouvost-Danon (Institut Pasteur). PMA, EDTA, EGTA,
pyrrolidine-dithiocarbamate (PDTC),
3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT),
DNP-lysine, and cycloheximide were purchased from Sigma (St. Louis,
MO). Actinomycin D and
N-tosyl-Phe-chloromethyl ketone (TPCK) were
obtained from Boehringer Mannheim (Mannheim, Germany).
Cell activation
RBL-2H3 cells were maintained as previously described (27). For
RNA preparations, one 75-cm2 flask (
2 x
107 cells) was used per experiment. Cells were sensitized
for 1 to 2 h in 20 ml of culture medium supplemented with 20 mM
HEPES and DNP-specific IgE as described above. After two washes with
PBS, cells were stimulated with DNP-BSA (50 or 100 ng/ml) in culture
medium prewarmed to 37°C. At each time point, samples were
immediately processed for RNA extractions (see below). For analysis of
nuclear transcription factor binding activity, cells were trypsinized
and resuspended at 2 x 106 cells/ml in complete
culture medium containing 20 mM HEPES and DNP-specific-IgE. After
1 h, suspension cells were washed and stimulated with DNP-BSA (50
or 100 ng/ml) at 37°C. Reactions were stopped by adding ice-cold PBS
before the preparation of nuclear protein extracts (see below). For
measurement of TNF- and ß-hexosaminidase secretion, adherent cells
(5 x 105 cells in 0.5 ml, 24-well plates) were
sensitized for 1 to 2 h with DNP-specific IgE in medium
supplemented with 20 mM HEPES (37°C). Cells were subsequently washed
with PBS and stimulated with DNP-BSA in complete medium at 37°C for
the indicated times. A sample of the medium (450 µl) was collected
from the wells, briefly centrifuged, aliquoted, and stored at -20°C
before testing.
RNA isolation and RNase protection assay
RNA isolation and RNase protection assays were conducted as
previously described (27). Rat TNF- cDNA (28) was obtained from Dr.
K. Decker (Biochemisches Institut, Universität Freiburg,
Freiburg, Germany). To develop antisense riboprobes of TNF- and
control ß-actin genes, nucleotides 199 to 433 of the TNF--coding
sequence covering exons 2 to 4 of the rat TNF- gene and nucleotides
2309 to 2460 in exon 3 of the rat ß-actin gene were amplified by PCR
using plasmid as a template and RT-PCR, respectively. The TNF- probe
yielded a protected fragment of 234 bp; the ß-actin probe yielded a
protected fragment of 151 bp. Hybridizations were conducted using 20 to
30 µg of total RNA, 500,000 cpm of the 32P-labeled
anti-sense rat TNF- riboprobe, and 50,000 cpm of the
32P-labeled antisense rat ß-actin riboprobe.
Nuclear extracts
All procedures were performed at 4°C. Nuclear extracts were prepared according to the method of Dignam et al. (29) with minor modifications. Briefly, 5 x 107 cells were washed in PBS and resuspended in HNB solution (15 mM Tris, pH 7.5, containing 0.5 M sucrose, 60 mM KCl, 0.25 mM EDTA, and 0.125 mM EGTA); then Nonidet P-40 was added to a final concentration of 0.2%. Cell lysis and integrity of nuclei were examined by light microscopy after addition of trypan blue. The cell homogenate was centrifuged for 10 min at 1,200 x g to pellet nuclei. The latter were resuspended in nuclear extraction buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 0.25 mM EDTA, and 0.37 M NaCl), and the released chromatin was pelleted by centrifugation at 50,000 x g for 30 min. Ammonium sulfate was added to a final concentration of 0.3 g/ml, and the precipitated proteins were centrifuged for 1 h at 100,000 x g. The protein pellet was resuspended in 100 µl of 20 mM HEPES, pH 7.9, containing 60 mM KCl, 20% glycerol, 0.25 mM EDTA, 0.125 mM EGTA, and protease inhibitors as previously described (29). The samples were dialyzed three times for 1 h each time in 300 ml of the same buffer. Insoluble material was removed by centrifuging for 3 min at 14,000 x g in a microcentrifuge. Soluble proteins were stored at -80°C in 30-µl aliquots; a 2-µl sample was reserved for determination of protein concentration by the Bradford assay (30).
Electrophoretic mobility shift assay (EMSA) and UV cross-linking
For oligonucleotide probes the following double-stranded
oligonucleotides were synthesized and used as labeled and nonlabeled
consensus probes in noncompetitive and competitive binding experiments
(only the sequence of one strand is shown, and consensus binding sites
are underlined; complementary probes were synthesized to give a 1-bp
overhang): 5'-GACAGAGGGGACTTTCCGAGAG-3' corresponding
to the NF-B binding site in the Ig -chain enhancer,
5'-GCGCTTGATGAGTCAGCCGGAA-3' containing an AP-1 binding
site, and 5'-GGGAGGAAAAACTGTTTCATACAGAAGGCGT-3'
corresponding to the distal NF-AT recognition site of the human IL-2
promoter. Double-stranded oligonucleotides corresponding to B
elements in the TNF- gene (see also Fig. 6
A) were the
following: B1, 5'-GACTCCGTGAATTCCCAGGAG-3'; B2,
5'-AGACAAGGGGGCTTTCCCTCCT-3'; B3,
5'-CGGAGAGGAGATTCCTTGATGC-3'; B4/NF-AT,
5'-TGAGATCATGGTTTTCTCCACC-3'; B 3',
5'-CCCTGGTCCTGGGAATTTCCCACTCTGG-3'; and CK2,
5'-GCTCTGGGGCTGCCCCATTCG-3'. Oligonucleotide AP-1 and
NF-B binding reactions were performed in a 20-µl volume containing
10 µg of the nuclear extract and 1 µg of poly(dI-dC) in binding
buffer (4% Ficoll, 20 mM HEPES (pH 7.9), 70 mM NaCl, 2 mM DTT, 0.01%
BSA, and 1 mM EDTA). The mixtures containing competitor oligonucleotide
(100x) or Abs were preincubated for 10 min at room temperature.
One nanogram of double-stranded oligonucleotides, which were end
labeled with [-32P]dATP and T4 polynucleotide kinase,
were added to the samples (sp. act., 100,000 cpm/ng) and allowed to
bind for 20 min at room temperature. Samples were loaded onto a 5%
nondenaturating polyacrylamide gel in 0.5 x Tris/borate/EDTA
buffer and allowed to migrate for 90 min at 200 V at room temperature.
Gels were dried on Whatman no. 3 MM filter paper (Clifton, NJ) and were
analyzed on a PhosphorImager.
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For UV cross-linking, 20 µg of nuclear extracts were incubated with a
radiolabeled bromodeoxyuridine (BrdUrd)-substituted double-stranded
oligonucleotide probes corresponding to the NF-B binding site in the
Ig -chain enhancer and the NF-B 3' site in the TNF-
gene (5'-GACAGAGGGGACTTTCCGAGAG-3',
5'-CCTCTCGGAAAGTCCCCTCTGT-3';
5'-CCCTGGTCCTGGGAATTTCCACTCTGG-3',
5'-TCCAGAGTGGGAAATTCCC-AGGACCAGG-3';
BrdUrd-substituted residues are in bold) and migrated as described
above. The wet gel was UV irradiated on a transilluminator (306 nm) for
15 min at room temperature and exposed under a PhosphorImager screen
for 15 min for localization of the shifted complexes. The complexes
were excised from the gel equilibrated in SDS sample buffer, resolved
on a 6 to 12% gradient SDS polyacrylamide gel, and analyzed on a
PhosphorImager.
CAT assays
A NF-B reporter construct carrying the tandem NF-B site of
HIV-1 as well as a control construct (31) were provided by Dr. M.
Körner. The rat TNF- promoter constructs (15) were gifts from
Dr. E. Beneveniste. For transfection, 4 x 106 cells
resuspended in complete medium were incubated with 10 µg of plasmid
DNA and electroporated using a gene pulser apparatus (Eurogentec,
Brussels, Belgium) at settings of 250 V and 1500 µFa and were plated
in 100-mm culture dishes. After 30 h IgE-sensitized cells were
stimulated for a period of 16 to 18 h with DNP-BSA (100 ng/ml).
Cells were scraped from dishes and lysed by freeze-thawing in 0.25 M
Tris, pH 8.0. CAT assays were conducted in duplicate as previously
described (15). The amount of CAT activity was normalized to the sample
protein, and values are expressed as the fold induction relative to
that in an unstimulated control.
Assay of ß-hexosaminidase and TNF- secretion
The release of mast cell mediators by exocytosis was monitored
using the ß-hexosaminidase assay as described previously (27). For
the TNF- bioactivity assay, based on the lysis of the target cells
(32), WEHI 164 clone 13 cells (3 x 104) in 50
µl of complete RPMI 1640 medium were plated in 96-well plates and
incubated at 37°C for 24 h. RBL-2H3 test supernatants (50 µl
of 3-fold serial dilutions in complete RPMI containing 2 µg/ml
actinomycin D) were added. After an overnight incubation, viable cells
were quantitated by use of the colorimetric MTT assay (33). Briefly, 25
µl of MTT (5 mg/ml) was added to the wells and incubated for 1
h. A 50-µl aliquot of the supernatant was discarded, and 100 µl of
solubilization solution was added. After overnight incubation OD was
measured at 570 nm. One unit of TNF- was defined as the dilution of
supernatant required to lyse 50% of WEHI 164 clone 13 target
cells. As a positive control for each experiment, serial dilutions of
known concentrations of recombinant murine TNF- (Genzyme, Cambridge,
MA) were used. To test the specificity of the cytotoxic effect, test
supernatants were preincubated with a 100-fold dilution of serum
specific to mouse TNF- (Genzyme) before incubation with the target
cells. Quantitation of secreted TNF- was also performed using an
ELISA kit specific for rat TNF- according to the manufacturer’s
instruction (BioSource, Camarillo, CA).
Statistical analysis of the data
A minimum of three experiments using independent cell cultures were performed for all reported data except when stated otherwise. The experiments shown are representative of each series of experiments or are a summary of the data from at least three experiments and are presented as the mean ± SEM. Statistical significance of the data was determined using Student’s paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA transcription in RBL-2H3 cells is independent of de
novo protein synthesis but requires continuous stimulation through
FcRI
IgE-sensitized RBL-2H3 cells were stimulated with various
concentrations of Ag, and TNF--mediated cytotoxicity toward the
TNF--sensitive WEHI 164 target cells was measured. Our
results shown in Figure 1A are
consistent with prior studies showing that TNF- is secreted by
RBL-2H3 cells in response to antigenic stimulation (7, 34, 35). Ag
concentrations in the range of 50 to 100 ng/ml of DNP-BSA appeared to
induce a maximal response. Unstimulated cells did not secrete TNF-,
since the values obtained were not significantly different from those
with medium alone. The cytotoxic effect of TNF- on the WEHI 164
cells was completely inhibited by preincubation of the stimulated cell
supernatants with an Ab specific for TNF- (data not shown). In
kinetic experiments (Fig. 1B) the secretion of TNF-
appeared as early as 30 min following stimulation; maximal levels were
achieved between 2 and 6 h and slowly declined thereafter.
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antisense riboprobes to measure TNF- steady
state mRNA levels in FcRI-stimulated RBL-2H3 cells. Figure 2A shows a representative
kinetic experiment. Unstimulated cells failed to induce TNF- mRNA
levels above the background control of transfer RNA. However,
aggregation of FcRI induced a rapid and easily detectable increase
in TNF- mRNA within 20 min. Maximum levels were achieved at
approximately 60 min of stimulation followed by a rapid decrease to the
background level of unstimulated cells within 180 min of stimulus
addition (Fig. 2, A and B). Quantitative analysis
showed that stimulation of the FcRI increased the steady state mRNA
levels by at least 15-fold (Fig. 2B).
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transcription required
continuous receptor aggregation, IgE -DNP-sensitized RBL-2H3 cells
were stimulated with DNP-BSA, and further aggregation of FcRI was
terminated at various time points by the addition of an excess of
hapten DNP-lysine. After incubation of cells for a total of 30 min, the
TNF- relative mRNA levels were determined by RNase protection assay.
As shown in Figure 3 TNF- mRNA steady
state levels were diminished in samples that had received hapten
compared with that in the sample that had been stimulated continuously
for 30 min. The decrease was more pronounced the earlier the hapten had
been added, clearly indicating that the induction of TNF- mRNA
expression can be promptly interrupted and requires ongoing receptor
stimulation.
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production can be regulated at multiple levels, including
at post-transcriptional and translational levels, we examined whether
the increase in steady state TNF- mRNA was due to de novo
transcription. Actinomycin D treatment at 2 µg/ml inhibited the
FcRI-mediated increase in TNF- mRNA by 92.9 ± 8.2% without
any significant effect on secretion of granule contents (4.0 ±
0.5%). These findings demonstrate that in RBL-2H3 cells,
FcRI-mediated TNF- secretion is largely dependent on the
synthesis of new TNF- mRNA. Treatment with cycloheximide (10
µg/ml), a potent inhibitor of protein synthesis, diminished TNF-
secretion by 97.0 ± 3.0%. Although the secretion of TNF- was
effectively inhibited, cycloheximide treatment did not abrogate the
synthesis of TNF- mRNA but instead increased the steady state levels
of this mRNA. Within 60 min TNF- mRNA expression reached more than
twice the level (253 ± 47.6%) in the absence of cycloheximide,
suggesting that synthesis of proteins is necessary for the rapid
down-regulation of TNF- gene expression. These results also
demonstrate that TNF- mRNA expression in RBL-2H3 cells is
independent of de novo protein synthesis and follows the rules of an
immediate early gene. Transcription factors described to mediate
immediate early gene transcription include NF-B and NF-AT. Consensus
sites for both these factors have been shown to be present in the
TNF- gene (see Fig. 6
A), and a role for NF-AT in TNF-
mRNA synthesis of mast cells has been proposed recently (19, 36). We
therefore focused on the examination of whether NF-B also plays a
role in the activation of TNF- gene transcription.
Induction of nuclear NF-B binding activity following FcRI
cross-linking
To examine whether antigenic stimulation of FcRI led to the
induction of nuclear NF-B DNA binding activity we employed a probe
corresponding to the consensus binding site for NF-B from the Ig
light chain enhancer. As a positive control we analyzed the
induction of nuclear AP-1 DNA binding activity that was recently
reported to occur in mast cells following antigenic stimulation (37).
Figure 4 shows that NF-B binding
activity was detected in nuclear extracts following stimulation of
FcRI. The protein-probe complex appeared as a broad band that could
be resolved to a doublet (upper complex and lower complex) in most
exposures (see also Fig. 6, B and C). Both bands
of the doublet were equally competed by an excess of unlabeled
oligonucleotides (Fig. 4A), whereas oligonucleotides
corresponding to the binding site for AP-1 did not compete for binding.
The AP-1 activity was detectable in unstimulated cells (Fig. 4B), and antigenic stimulation led to increased AP-1
binding activity, which was inhibited with excess unlabeled AP-1
consensus binding site oligonucleotides, but not with oligonucleotides
corresponding to the NF-B site (Fig. 4B).
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B activity following antigenic stimulation. As shown in Figure 5A no significant NF-B
binding activity was detected in unstimulated cells. Following exposure
to Ag, NF-B binding activity could be detected as early as 15 min
(data not shown) and reached maximal activity at approximately 60 min
of stimulation (Fig. 5A). Continued stimulation for 150 min
resulted in decreased NF-B binding activity to levels similar to
those found in unstimulated cells. Quantitative analysis showed a
maximal induction of 6- to 16-fold for NF-B binding activity in the
nucleus in five independent experiments (Fig. 5B). In
comparison, the AP-1 binding activity present in unstimulated cells
also increased, with a maximal response at approximately 60 min of
stimulation (Fig. 5A). However, unlike NF-B, AP-1 binding
activity was stable for a prolonged period of time, with elevated
levels maintained at 150 min of stimulation. Quantitative analysis of
the induced levels of AP-1 binding activity showed an increase of 3- to
4-fold (Fig. 5B). The requirement for ongoing receptor
stimulation was also examined. Figure 5C shows that when
receptor aggregation was terminated by adding an excess of hapten at
early time points AP-1 binding activity was not altered, suggesting
that a short stimulus was sufficient to induce maximal binding activity
even in the absence of stimulation (Fig. 5C). In contrast,
NF-B binding activity was rapidly reversed, indicating that NF-B
binding activity depends, like TNF- mRNA production, on the
continuous stimulation through FcRI.
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B consensus binding sites in transcriptional
induction of the TNF- gene
To investigate whether the induced NF-B binding activity could
play a role in TNF- gene transcription we examined whether the
various B sites present in the TNF- gene could also bind the
induced NF-B binding complexes. As a first approach we tested the
different individual sites present within the TNF- gene (Fig. 6A) for their capacity to
inhibit formation of the protein-probe complex. The results given in
Figure 6B show that besides the consensus site
double-stranded oligonucleotide, which was the most powerful
inhibitor, all other oligonucleotides inhibited complex formation to
some degree. Differences in the capacity to inhibit the upper and lower
complexes were noted. The oligonucleotides corresponding to the
B4/NF-AT and B3' sites substantially inhibited both lower
and upper complexes, whereas the other B as well as B pseudosite
CK2 oligonucleotides preferentially inhibited the upper complex. This
was further confirmed when the oligonucleotide probes were directly
tested for their binding capacities (Fig. 6C).
Interestingly, in these experiments we could not detect significant
binding with oligonucleotide probes corresponding to B1 and B3
sites for the upper complex and B4/NF-AT for the lower complex,
suggesting that the interaction with these sites may be of low
affinity.
To functionally test the role of the inducible NF-B binding activity
we used NF-B and TNF- promoter constructs encoding CAT as a
reporter gene. In the first set of experiments we tested a promoter
construct containing the tandem NF-B site of the HIV-1 promoter as
well as a control construct where both these sites have been deleted
(see Fig. 7A). As shown in
Figure 7B stimulation of RBL-2H3 cells transfected with this
construct readily led to an induction of CAT activity. No activity was
induced with the control promoter construct. These results indicate
that stimulation of FcRI in RBL-2H3 cells can induce
NF-B-dependent transcription of a heterologous promoter. We next
examined the effect of stimulation on a TNF- promoter construct
containing all 5' B sites as well as a promoter construct containing
only the most proximal B4/NF-AT site. The latter site was shown to
bind the transcription factor NF-AT and to play a role in the induction
of TNF- in a variety of cells (21, 36). A construct in which the
entire promoter sequence was inserted in a reverse orientation was used
as a control. As shown in Figure 7B, following stimulation
of transfected RBL-2H3 cells CAT activity could be induced in
constructs containing the full promoter. No significant induction was
detectable in constructs in which three B sites, but not the
proximal B4/NF-AT site, were deleted.
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B in FcRI-dependent
transcription we used an alternative approach based on the use of
pharmacologic inhibitors. This allowed the study of FcRI-dependent
transcription of TNF- mRNA at the level of the gene in its native
chromatin structure. In the first series of experiments we used a
potent inhibitor of the nuclear localization of NF-B-related
factors, the antioxidant PDTC (38, 39, 40). RBL-2H3 cells pretreated or not
with PDTC were subsequently stimulated with Ag, and the effects on
TNF- mRNA expression and nuclear localization of several
transcription factors were examined. For the latter we particularly
focused on the nuclear DNA binding activities of NF-B, AP-1, and
NF-AT. As revealed in Figure 8 TNF-
mRNA expression (Fig. 8B) was dramatically inhibited at
concentrations where NF-B binding activity was also substantially
inhibited (Fig. 8A). No significant effect was observed on
the binding activities of AP-1 and NF-AT in the presence of PDTC (Fig. 8A). The latter binding activity appeared as several bands
that were inhibited with an excess of unlabeled oligonucleotides (data
not shown). In addition, we examined the effect of the protease
inhibitor TPCK, which has also been shown to be a powerful inhibitor of
NF-B (41). As shown in Figure 8B, in RBL-2H3 cells this
compound inhibited TNF- mRNA steady state levels in a dose-dependent
manner as well as the activation of NF-B nuclear binding activity.
The binding activity of the transcription factor AP-1 was not
significantly affected, while that of NF-AT was enhanced in the
presence of TPCK (Fig. 8A).
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B binding complexes
To determine the composition of the related NF-B complexes we
initially tested, using supershift analysis, whether the p50 (NF-B1)
and p65 (Rel-A) proteins might be present in the binding complexes,
since these are the most abundant NF-B protein components in various
cells (42). Whereas both Abs to p50 and p65 readily led to a supershift
of NF-B binding proteins present in nuclear extracts of
LPS-stimulated rat spleen cells (Fig. 9A), no supershift was
observed with the complex present in nuclear extracts from RBL-2H3
cells. Two other Abs to p50 and p65 directed toward epitopes that
differed from the above Abs also did not induce a supershift (data not
shown). In addition, as shown in Figure 9A, the shifted
complexes in RBL-2H3 mast cells migrated with slower mobility than the
classical NF-B complex present in LPS-stimulated rat splenocytes.
Therefore, we tested Abs directed to related NF-B proteins such as
Rel-B, Rel-C, p52, and an Ab directed to a conserved region in NF-AT
proteins that has been shown to bind to NF-B elements under certain
conditions (43). However, none of these Abs had an effect on the
relative mobility of the original complex (data not shown). These
results suggested that the induced NF-B binding activity is not
likely to be comprised of the classical NF-B binding proteins. This
was further investigated by assessing the molecular mass of the protein
adducts using UV cross-linking experiments with a BrdUrd-substituted
consensus B probe. In preliminary experiments we verified that the
presence of BrdUrd in the probes did not affect the migration of the
complexes. Cross-linking was performed by UV irradiating the native
acrylamide gel. Due to the close migration of the UV cross-linked upper
and lower complexes in acrylamide gels we excised both complexes
together and resolved the components by SDS-PAGE. As shown in Figure 9B, cross-linking revealed the presence of three species
with molecular masses of 90, 100, and 110 kDa. The same pattern was
obtained with the 3' B probe corresponding to the site found in the
3'-flanking region of the TNF- gene. Cross-linked samples from
LPS-stimulated rat splenocytes did not reveal proteins with the
above-mentioned molecular mass (data not shown). Furthermore, the size
of the identified protein adducts did not correspond to the molecular
mass of previously described protein adducts of p50, p65 (Rel-A), or
Rel-C (44).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression has been shown to be tightly regulated and
to involve an array of different transcription factors whose activity
depends largely on the stimulus as well as on the cell type. Regulation
of TNF- gene expression in mast cells is of particular interest,
since in these cells, TNF- is released as a prestored component of
cytoplasmic granules but is also newly synthesized upon cell
activation. The newly synthesized TNF- accounts for the sustained
release of the cytokine. Our study with RBL-2H3 cells as well as a
previous study (2) show that the new synthesis of TNF- gene
transcripts follows the rules of an immediate early gene and does not
require de novo synthesis. Transcription factors such as NF-AT and
NF-B are likely candidates for the regulation of immediate early
genes, and consensus binding sites for both these transcription factors
are present in the TNF- gene (16, 21). Recent evidence implicates
the transcription factor NF-AT in the expression of newly synthesized
TNF- in PMA/ionophore- or FcRI-stimulated CPII mast cells (19, 36). The results of these studies are consistent with NF-AT regulation
of the TNF- gene observed in human B and T lymphocytes, where the
most proximal human B promoter element was reported to bind NF-AT
and drive reporter-dependent gene expression following stimulation (20, 21). However, species-specific regulation of the TNF- gene is
suggested by the fact that this binding site is not completely
conserved among the mouse, rat, and human (14, 21). In addition,
differences in the differentiation stage and tissue origin of the
cell may influence how the genes are regulated. For example, in CPII
cells induction of NF-B binding activity was not observed, although
it was detected in MC/9 mast cells by NF-B-dependent reporter assays
(45) and in Cl.MC/C57.1 mast cells by EMSAs (46).
To reexamine the relationship among FcRI stimulation, TNF- gene
expression, and nuclear localization of transcription factor complexes
we used RBL-2H3 cells as a model system. Our findings reveal a novel
and previously uncharacterized NF-B-like binding activity as
important to transient activation of the TNF- gene in response to
aggregation of FcRI. These results provide a candidate activity that
may explain the apparent discrepancies in the above-mentioned studies
on different mast cell lines. Several independent approaches
established the relationship between TNF- mRNA expression and
induction of this novel nuclear NF-B binding activity in RBL-2H3
cells: 1) the analysis of B elements present in the TNF- gene
loci showed the presence of several elements that have the capacity to
inhibit binding and/or bind directly the nuclear complex; 2) both
TNF- mRNA expression and nuclear NF-B binding activity were
transiently induced with similar kinetics, and both required continuous
receptor stimulation; 3) analysis of AP-1, NF-AT, and NF-B nuclear
binding activities in the presence of pharmacologic agents that inhibit
nuclear activation of the described NF-B binding activity revealed
that the specific loss of NF-B binding activity also resulted in a
loss of TNF- mRNA expression; and 4) deletion of 5' B elements
present in the TNF- promoter (except the one most proximal to the
initiation site) abolished all measurable FcRI-dependent induction
in TNF- promoter-reporter assays.
An interesting feature of the induced NF-B binding activity in
RBL-2H3 cells was the rapid and transient nature of the response, which
was remarkably similar to the kinetics of TNF- mRNA induction. In
general, it is thought that NF-B binding activity is relatively
stable (42); however, a similar rapid and transient response has been
shown in murine pre B (70Z/3) and lymphoma (EL4) cell lines when
stimulated via the type I IL-1R (47). We also found that interruption
of receptor aggregation with hapten resulted in a rapid reversal of
both nuclear NF-B binding activity and TNF- gene expression,
suggesting that both processes can be rapidly modulated and are
dependent on continuous stimulation. This is consistent with a previous
report (48) of the sustained NF-B binding activity as being
dependent on the continuous presence of the external stimulus. In
contrast, we demonstrated that AP-1 activity is not reversed by
abrogating the formation of new receptor aggregates. In a similar
manner, mRNA expression of the immediate early gene c-fos
after a short stimulus could not be terminated or reversed by the
interruption of receptor aggregate formation (49).
NF-B-dependent expression of TNF- has been reported in a number
of systems. These include LPS-stimulated macrophages and monocytes,
where increased TNF- production was found to correlate with
increased NF-B binding activity (16, 17, 38). NF-B-mediated
induction in murine macrophages and rat astrocytes has also been
defined to function through B elements found in the 3'-flanking
region of the TNF- gene downstream of the polyadenylation site (13, 15). In our study the oligonucleotide corresponding to the 3'-flanking
region of the TNF- gene strongly inhibited formation of both upper
and lower nuclear complexes observed in RBL-2H3 cells and could itself
induce a shift of these complexes. Thus, the absence of the 3' B
elements from the TNF- promoter-CAT construct probably explains the
lower transcriptional activity observed with this reporter compared
with that with the NF-B reporter construct. However, a direct test
of this hypothesis would require mutational analysis of the 3' B
elements in the native gene.
Molecular analysis of the induced NF-B complexes suggests that they
are comprised of novel and of as yet uncharacterized proteins by
several criteria: 1) the observed complexes migrated with a lower
electrophoretic mobility than the classical NF-B binding activity of
LPS-stimulated rat splenocytes, which were shown to contain both p50
and p65; 2) using a series of Abs we did not detect the presence of
p50, p65, Rel-B, Rel-C, or p52 in the complex; and 3) molecular size
analysis using UV cross-linking experiments revealed proteins with
molecular masses of 90 to 110 kDa. These proteins were not detected
with the NF-B complex from LPS-stimulated rat splenocytes. These
results suggest that in addition to the classical NF-B binding
complexes implicated in TNF- gene expression we have identified
novel NF-B binding proteins that also regulate the induction of
TNF- mRNA. Novel NF-B-like complexes have been reported in other
systems and include a p50-related molecule of 55 kDa and a complex of
proteins, with a molecular mass of 110 to 115 kDa, that are expressed
in the brain during development (50, 51). The molecular mass of the
latter is similar to that observed in the RBL mast cell model. Whether
these proteins are identical with those we describe awaits
verification.
|
Acknowledgments |
|---|
promoter-CAT
reporter construct; and Drs. J. Dastych, M.
Benhamou, and R. Monteiro for critically reading the manuscript. We
are grateful to Dr. M. Körner and our colleagues in the mast cell
signal transduction laboratory for motivating discussions. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ulrich Blank, Unité Immuno-Allergie, Institut Pasteur, 25–28 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address:
3 Abbreviations used in this paper: FcRI, high affinity IgE receptor; NF-AT, nuclear factor of activated T cells; AP-1, activator protein-1; RBL-2H3, rat basophilic leukemia 2H3 cell line; PDTC, pyrrolidine dithiocarbamate; MTT, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; TPCK, N-tosyl-Phe-chloromethyl ketone; EMSA, electrophoretic mobility shift assay; BrdUrd, bromodeoxyuridine; CAT, chloramphenicol acetyltransferase.
Received for publication March 30, 1998. Accepted for publication June 25, 1998.
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