|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4318-4327
Interleukin-9 Regulates NF- B Activity Through BCL3 Gene
Induction
By
Mélisande Richard,
Jamila Louahed,
Jean-Baptiste Demoulin, and
Jean-Christophe Renauld
From the Ludwig Institute for Cancer Research, Brussels Branch,
Brussels, Belgium; and the Experimental Medicine Unit, Christian de
Duve Institute of Cellular Pathology, Université Catholique de
Louvain, Brussels, Belgium.
 |
ABSTRACT |
BCL3 encodes a protein with close homology to I B proteins
and interacts with p50 NF-B homodimers. However, the regulation and
transcriptional activity of BCL3 remain ill-defined. We observed here
that interleukin-9 (IL-9) and IL-4, but not IL-2 or IL-3, transcriptionally upregulated BCL3 expression in T cells and
mast cells. BCL3 induction by IL-9 was detected as soon as 4 hours after stimulation and appeared to be dependent on the Jak/STAT pathway. IL-9 stimulation was associated with an increase in p50 homodimers DNA binding activity, which was mimicked by stable BCL3 expression. This contrasts with tumor necrosis factor
(TNF)-dependent NF-B activation, which occurs earlier, involves
p65/p50 dimers, and is dependent on IB degradation. Moreover, IL-9
stimulation or BCL3 transient transfection similarly inhibited
NF-B-mediated transcription in response to TNF. Taken together, our
observations show a new regulatory pathway for the NF-B
transcription factors through STAT-dependent upregulation of
BCL3 gene expression.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE BCL3 GENE WAS originally
identified at the junction in the t(14;19) chromosome translocation in
certain cases of human B-cell chronic lymphocytic
leukemias.1 In leukemic cells with this translocation,
expression of this gene is constitutive, pointing to a role for
BCL3 in leukemogenesis,1 although there is no
experimental evidence as yet for a role of BCL3 in
transformation. BCL3 mRNA is detected in different tissues,
especially in spleen and other lymphoid organs.2
BCL3 null mutant mice develop normally but exhibit severe
defects in cellular and humoral immune responses to pathogens and show
altered spleen micro-architecture associated with a loss of splenic B
cells.3,4 Although these observations show that BCL3 is
involved in the regulation of immune responses, its precise role is
still unknown.
BCL3 belongs to the IB family of proteins, featuring 7 tandem
ankyrin repeats that are similar to those found in
p105/NF-B15 and other IB members: p100/NF-B2,
IB , IB , and IB (for review see Baldwin6
and Baeuerle and Baltimore7). Apart from this conserved
domain, BCL3 also features a Pro-rich amino-terminal domain and a Pro-
and Ser-rich carboxy-terminal domain.1
Like other IB proteins, BCL3 interacts with NF-B transcription
factors via its ankyrin repeats. NF-B consists of dimers of subunits
that contain a conserved Rel homology domain required for dimerization,
interaction with IB, and DNA binding. The NF-B family can be
divided into two groups: (1) c-Rel, p65/RelA, and RelB, which contain a
well-defined transactivation domain; and (2) p50 and p52, which are
generated by proteolytic processing from their precursors,
p105/NF-B1 and p100/NF-B2, respectively. The major form of
NF-B, in most cases, is a heterodimer of the p50 and p65 subunits.
The classical pathway leading to NF-B activation has been
extensively reviewed.8,9 p65/p50 dimers are sequestrated in
the cytoplasm by interaction with IB proteins and translocated into
the nucleus in response to various extracellular stimuli that induce
IB phosphorylation and degradation. This results in B-dependent
transcription of a variety of genes implicated in stress and immune responses.
Contrasting with IB and IB, which specifically interact
with dimers containing p65 or c-Rel,10,11 BCL3 specifically interacts with p50 or p52 homodimers.12,13 Moreover, BCL3
is predominantly a nuclear protein,2,14 whereas IB is
mainly a cytoplasmic protein.14,15
Whether BCL3 acts as a bona fide IB molecule has been disputed.
Initial reports indicated that BCL3 can inhibit p50 homodimers DNA
binding.12,16 However, Fujita et al17 showed
that BCL3 can form a ternary complex on DNA in association with p50
homodimers. Recently, it was clearly demonstrated that BCL3 favors p50
dimer formation by recruiting p50 monomers from the cytoplasmic pool of
p105/p50 dimers and enhances nuclear translocation and DNA binding of
p50 dimers.18
The precise role of p50 homodimers is still a matter of controversy:
some investigators have reported that they are unable to activate
transcription,12,19 whereas others have reported that p50
homodimers can activate a promoter containing B
sites.20,21 In this context, BCL3 was proposed to be an
activator of transcription, either by removing p50 homodimers from B
sites and allowing p65/p50 transactivating complexes to target
DNA12 or by coactivation through p50
homodimers.17 Other observations suggest that BCL3 inhibits
B-dependent transcription.22
The regulation of BCL3 expression has been poorly investigated
so far. This gene was shown to be induced by mitogenic stimuli in B and
T cells.1,23 The role of cytokines in this process has not
been studied yet. Interleukin-9 (IL-9) is a multifunctional cytokine
secreted by activated T cells24 and exerts its effects on a
variety of cell types, especially in the immune system (mast cells, B
and T lymphocytes, hematopoietic progenitors, and
eosinophils).25 Interestingly, IL-9 was described as an
antiapoptotic factor for thymic lymphomas,26 and transgenic
mice expressing the IL-9 protein at a high level are more susceptible
to lymphoma development than control littermates.27 More
recently, a role for IL-9 has been suggested in allergy and
asthma.28
We report here that IL-9 induces the expression of BCL3 mRNA
and protein and enhances the DNA binding of p50 homodimers. Moreover, BCL3 induction by IL-9 appears to be STAT-dependent and leads to
repression of tumor necrosis factor (TNF)-induced NF-B
transactivation. Our results point towards a previously undocumented
pathway leading to NF-B regulation by cytokines independently of
IB degradation.
| |
MATERIALS AND METHODS |
Cell culture, cell transfections, and cytokines.
T-helper cell clones TS2 and TS329 were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal calf serum (FCS), 50 µmol/L 2-mercaptoethanol, 0.55 mmol/L L-arginine, 0.24 mmol/L L-asparagine, and 1.25 mmol/L
L-glutamine. These factor-dependent cell lines were able to grow in the
presence of either IL-2, IL-4, or IL-9 without antigen and feeder
cells.24,29 ST2K9 (a gift from Dr E. Schmitt,
Gütenberg University, Mainz, Germany) was cultured in the same
medium. BW5147, a murine lymphoma cell line (ATCC, Rockville, MD), and
MC9p3, an autonomous variant of the MC9 mast cell line (a gift from Dr
C. Petit-Frère, Institut Henri Beaufour, Les Ullys, France), were
cultured in Iscove-Dulbecco's medium supplemented with 10% FCS, 50 µmol/L 2-mercaptoethanol, 0.55 mmol/L L-arginine, 0.24 mmol/L
L-asparagine, and 1.25 mmol/L L-glutamine. L138 (a gift from Dr L. Hültner, GSF, München, Germany) and MC9, two mast cell
lines, were cultured in the same medium in the presence of IL-3 or
IL-9. Mouse bone marrow-derived mast cells (BMMC) were obtained by
culturing bone marrow from Balb/c mice for 4 weeks in enriched medium
(RPMI-1640 containing 0.55 mmol/L L-arginine, 0.24 mmol/L L-asparagine,
1.25 mmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, and 20% FCS)
supplemented with either recombinant murine IL-3 (20 U/mL) or a
combination of IL-3 (20 U/mL) and IL-9 (200 U/mL).
Fluorescence-activated cell sorting (FACS) analysis of this population
showed an homogenous staining by biotinylated IgE and no staining with
anti-Mac1, anti-Mac2, anti-Mac3, and anti-Thy1 antibodies.
For stable transfection of a BCL3 expression plasmid, the
BCL3 cDNA was cloned in the pEF-BOS plasmid30 in
which a puromycin resistance gene has been inserted for selection of
transfectants. Cells were transfected by electroporation (1,500 µF,
74 ohm, and 290 V for MC9p3; 1,500 µF, 74 ohm, and 230 V for BW5147)
with 40 µg of sterile DNA. Clones and pools of transfected cells were selected using puromycin (1.8 µg/mL), and BCL3 expression was checked
by Western blot analysis with an affinity-purified rabbit polyclonal
antibody directed against the C-terminal part of BCL3 (Santa
Cruz-sc#185; Santa Cruz Laboratories, Santa Cruz, CA; 1 µg/mL). BW5147 cells expressing wild-type or mutated hIL-9R were obtained as previously described31 and cultured in the
presence of 1.5 µg/mL puromycin. Two additional hIL-9R mutants, mut6
(activating STAT1 and 3) and mut7 (activating STAT5), were similarly
transfected in BW5147 (Demoulin et al, manuscript in
preparation). In the mut6 hIL-9R, mutation of leucine 368 into arginine reduced STAT5 activation to less than 5% of the
activation observed with the wild-type hIL-9R, without affecting the
activation of other STATs. In the mut7 hIL-9R, replacement of glutamine
370 by a leucine abolished STAT3 and STAT1 activation, without
affecting that of STAT5.
Recombinant human IL-9 (2 × 107 U/mg), mouse IL-4
(3.8 × 106 U/mg), mouse IL-6 (109 U/mg),
and mouse IL-9 (5 × 107 U/mg) were produced in the
baculovirus system in our laboratory and purified as previously
described.32 Human recombinant IL-2 (3.5 × 106 U/mg) was provided by Cetus (Chiron Corp,
Amsterdam, The Netherlands) and mouse recombinant IL-3 (2 × 107 U/mg) was a gift of Dr J.Y. Bonnefoy (Glaxo, Geneva,
Switzerland). Human recombinant TNF was supplied by
Peprotech (Rocky Hill, NJ). Actinomycin D (ICN, Irvine,
CA), protein synthesis inhibitor cycloheximide (Sigma, St
Louis, MO), and proteasome inhibitor MG-132
(Biomol, Plymouth Meeting, PA) were used at 5, 10, and 50 µmol/L, respectively.
Subtractive hybridization.
Total RNA was prepared from TS2 cells stimulated with IL-2 (200 U/mL)
or mIL-9 (200 U/mL) for 48 hours, using guanidium isothiocyanate lysis
and CsCl gradient centrifugation.33 Polyadenylated RNA was
purified from total RNA with oligo(dT) cellulose columns (Pharmacia, Uppsala, Sweden). Double-stranded cDNA was generated from
5 µg polyA+ RNA using an oligo(dT) primer and the
SuperScript Choice System for cDNA synthesis, according to the
manufacturer's recommendations (GIBCO BRL, Grand Island,
NY). Representational difference analysis was performed
as described.34
After 3 rounds of subtraction, final difference products were digested
with Dpn II and cloned into the BamHI site of pTZ19R. Double-stranded plasmid DNA was prepared and sequenced with a Thermo-sequenase Sequencing kit (Amersham, Arlington Heights, IL).
Sequence comparisons with the GenBank and EMBL databases were performed
with the BLAST search program (NCBI, Bethesda, MD).
Oligo(dT)-primed cDNA libraries generated from IL-9-stimulated TS2
cells were screened with the mouse BCL3 Dpn II fragment.
Preparation of mRNA and Northern blot.
Total RNA was prepared using guanidium isothiocyanate lysis and CsCl
gradient centrifugation from TS2, TS3, ST2K9, L138, MC9, and BMMC.
These IL-9-responsive cell lines were cultured in medium containing
saturating concentrations of the indicated cytokines as follows: 10 days in the presence of 100 U/mL IL-2 or 200 U/mL IL-9 (or IL-4) for
TS2, TS3, and ST2K9 cells; 10 days in the presence of 200 U/mL IL-3 or
200 U/mL IL-9 for L138 and MC9 cells; and 28 days in the presence of 20 U/mL IL-3 or 20 U/mL IL-3 and 200 U/mL IL-9 for BMMC. BW5147 was
cultured for 2 days with or without 500 U/mL IL-9 or IL-4 or 10,000 U/mL IL-6. Total RNA was extracted from BW5147 cells transfected with
mutants of the human IL-9 receptor and stimulated for 24 hours with 500 U/mL human or murine IL-9 using Trizol reagent (GIBCO BRL), according
to the manufacturer's guide.
Ten micrograms of total RNA was fractionated by electrophoresis in a
1.3% agarose gel containing 2.2 mol/L formaldehyde and were
transferred onto a Hybond-C Extra nitrocellulose membrane (Amersham).
cDNAs were labeled using the Rediprime DNA labeling kit from Amersham.
Hybridizations and washes were performed as described.35
The BCL3 probe was a 1.8-kb cDNA containing the complete coding
sequence. After autoradiography, all blots were reprobed with a chicken
-actin probe to control for even loading of RNA.
Reverse transcription-polymerase chain reaction
(RT-PCR) analysis and Southern blotting.
Reverse transcription was performed on 5 µg Trizol-purified total RNA
with an oligo(dT) primer. cDNA corresponding to 20 ng of total RNA was
amplified for 25 cycles by PCR with specific primers for murine
BCL3 as follows: sense 5'-GCGCAGCGGCTGCGACGT-3' (from position 204 of the cDNA sequence; GenBank accession no. AF067774) and antisense 5'-CATCCGTCTCAGCTGCTTCCT-3' (from
position 1394 of the cDNA sequence); and for -actin: sense
5'-ATGGATGACGATATCGCTGC-3' and antisense
5'-GCTGGAAGGTGGACAGTGAG-3' (20 cycles). The post-PCR products were analyzed in ethidium bromide-stained 1% agarose gel.
Specific amplification was confirmed after blotting (Zeta-Probe membrane; Bio-Rad, Hercules, CA) by hybridization with
internal radioactive probes. The internal probe was
5'-GTCCACGACATCATGCTACTG-3' for -actin and
5'-TGTGGTGATGACAGCCAGGTG-3' for the mouse BCL3. Radioactive signals were quantified by Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and the
BCL3/actin ratios were calculated.
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts were prepared as described,36 with minor
modifications. Briefly, cells (1.5 × 107) were
stimulated for the indicated periods with IL-9 (100 U/mL) or TNF (10 ng/mL), washed with phosphate-buffered saline (PBS), and resuspended in
1 mL ice-cold hypotonic buffer A for 15 minutes (10 mmol/L HEPES
buffer, pH 7.5, containing 10 mmol/L KCl, 1 mmol/L MgCl2,
5% glycerol, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 0.5 mmol/L dithiothreitol, 1 mmol/L Pefabloc [Boehringer Mannheim, Mannheim, Germany], 10 µg/mL aprotinin, 1 mmol/L
Na3VO4, and 5 mmol/L NaF). The cells were lyzed
by adding 65 µL NP-40 10% and vortexing. Nuclei were pelleted (30 seconds at 14,000 rpm) and extracted for 30 minutes in 100 µL
hypertonic buffer B (buffer A supplemented with HEPES [20 mmol/L],
glycerol [20%], and NaCl [420 mmol/L]). Nuclear debris were
removed through 2 minutes of centrifugation. Analysis of DNA binding
activity was performed as described37 using
32P-labeled oligonucleotide probes corresponding to (1) a
palindromic B motif38: upper strand,
5'-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA-3', and bottom strand,
5'-GATCTAAGGAGAGGGGAATTCCCCTGCCGTTG-3'; and (2) an Sp1 DNA
binding motif39: upper strand,
5'-ATTCGATCGGGGCGGGGCGAGC-3', and bottom strand,
5'-ATGCTCGCCCCGCCCCGATCGA-3'.
Supershifts were performed by adding antibodies to the incubating
mixture of nuclear extracts and labeled DNA probe. Four micrograms of
anti-p50 antibody (sc#1192-X; Santa Cruz) and 2 µg of anti-p65
antibody (sc#372-X; Santa Cruz) were used per lane.
Western blot.
IB phosphorylation was assayed using the PhosphoPlus
IB(Ser32) Antibody Kit (New England Biolabs, Beverley,
MA). Briefly, cells (2 × 106) were
stimulated with IL-9 (100 U/mL) or TNF (10 ng/mL) for 5, 10, or 30 minutes and lyzed in 100 µL SDS Sample Buffer (Bio-Rad). Proteins
were fractionated on precast Novex sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE;
8%) and electrophoretically transferred to a polyvinylidene difluoride
(PVDF) membrane (Amersham). Membranes were blocked in 5%
nonfat dry milk, washed, and probed with diluted (1/1,000)
affinity-purified rabbit polyclonal antibodies (directed against
IB or the phosphorylated Ser32 of this protein) and with
horseradish peroxidase-linked antirabbit antibody (1/2,000). An ECL
detection kit (Phototope-HRP Western Detection Kit; New England
Biolabs) was used for expression of chemiluminescence.
For BCL3 detection, BW5147 cells (2 × 107) were
stimulated or not stimulated with IL-9 (100 U/mL) for different periods
of time. Total cell extracts were prepared by lysis in RIPA buffer (PBS, pH 7.4, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS). Fifty micrograms of proteins was loaded with 1 vol of SDS Sample Buffer
(Bio-Rad) on a precast Novex SDS-PAGE polyacrylamide gel (8%). After
transfer, the PVDF membrane (Amersham) was blocked in 5% nonfat dry
milk, washed, and probed with an affinity-purified rabbit polyclonal
antibody directed against the C-terminal part of BCL3 (sc#185; Santa
Cruz; 2 µg/mL) and with horseradish peroxidase-linked antirabbit
antibody (1/5,000; Transduction Laboratories, Lexington, KY). An ECL detection kit (Phototope-HRP Western
Detection Kit; New England Biolabs) was used for expression of chemiluminescence.
Luciferase activity assay.
The reporter plasmid (pGL3-TK-BPD) was obtained by subcloning two
palindromic NF-B binding sites (5' GGGGAATTCCCC 3') and the TK promoter into pGL3 basic (Promega, Madison, WI)
upstream of the firefly luciferase coding sequence. As an internal
control, we used the pRL-TK vector (Promega) containing the
Renilla luciferase gene under the control of the TK promoter.
For BW5147 cells, 10 million cells were coelectroporated with 40 µg
pGL3-TK-BPD and 1 µg pRL-TK (230 V, 74  , and 1,500 µF). The
pool of transfected cells was divided into four equal fractions, and
each fraction was stimulated either with mIL-9 (100 U/mL), mIL-4 (500 U/mL), hTNF (10 ng/mL), or a combination of TNF and IL-9 or IL-4
or was left unstimulated. After 8 or 48 hours, cells were pelleted and
lyzed. Both luciferase activities were monitored with the Dual-Luciferase Reporter Assay System kit (Promega) according to the
manufacturer's instructions. For COS cells, 5 × 105
cells per well were seeded in 6-well plates 24 hours before
transfection with Lipofectamin (GIBCO BRL). Cells were transfected with
250 ng pGL3-TK-BPD, 250 ng pRL-TK, and either 1 µg of the
BCL3 cDNA in the pEF-BOS plasmid or 1 µg of the empty vector.
After 24 hours, cells were stimulated or not stimulated with TNF (10 ng/mL). Luciferase activities were monitored 8 hours later as described above.
| |
RESULTS |
IL-9 induces BCL3 gene expression in mouse T lymphocytes and mast
cells.
To find a specific gene expression pattern that could explain the
effects of IL-9 on mouse T lymphocytes, we performed a representational difference analysis of gene expression on TS2 cells. These T-helper cells were cultured either with IL-2 or IL-9 for 10 days.
Poly-A+ RNA was extracted and the respective
oligo(dT)-primed cDNAs were prepared, digested with Dpn II, and
then used to create both amplicons. After three rounds of subtractive
hybridization, the third difference product was cloned and 57 clones
were sequenced. Five independent IL-9-specific cDNA sequences were
identified and one of them was found to match the sequence of
BCL3.
Full-length cDNA clones obtained from a TS2 cDNA library were sequenced
and found to differ from the published BCL3 gene23 in 23 nucleotides, resulting in 11 amino acid changes. Because the same
amino acid changes were found in a cDNA library from another unrelated
cell line (BW5147 from AKR mice) and because we have never identified
any cDNA that matches the published sequence, we assume that these
clones correspond to the bona fide BCL3 gene and not to
a new BCL3-related gene (GenBank accession no. AF067774).
To confirm BCL3 mRNA expression upon IL-9 stimulation, we
performed a Northern blot hybridization with RNA from T-helper cell clones that can grow in the presence of either IL-2, IL-4, or IL-9
(TS2, TS3, or ST2K9). As shown in Fig 1, a
strong BCL3 signal was detected in all T-cell clones stimulated
with IL-9 but not when the cells were cultured in the presence of IL-2.
Interestingly, both IL-9 and IL-4 induced the BCL3 mRNA in TS2
cells as well as in the BW5147 thymic lymphoma. We observed a similar
BCL3 gene induction by IL-9 in primary BMMC and in mast cell
lines, such as MC9 and L138, that can grow either with IL-3 or IL-9.
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
IL-9 induces BCL3 expression. The indicated
IL-9-responsive cells were cultured in medium containing saturating
concentrations of the indicated cytokines: 10 days in the presence of
100 U/mL IL-2, 200 U/mL IL-4, or 200 U/mL IL-9 for T-helper cell clones
(TS2, TS3, and ST2K9) and 10 days in the presence of 200 U/mL IL-3 or
200 U/mL IL-9 for mast cell lines (L138 and MC9). BMMC were cultured in
the presence of 20 U/mL IL-3 or a combination of IL-3 and IL-9 (200 U/mL). The mouse lymphoma line BW5147 was stimulated for 2 days with
500 U/mL IL-9 or IL-4 or 10,000 U/mL IL-6. After electrophoresis of 10 µg of total RNA and transfer to nitrocellulose, filters were
hybridized with a 32P-labeled mouse BCL3 cDNA
probe. Hybridization with a -actin probe confirmed that
comparable amounts of RNA had been loaded in each lane.
|
|
The kinetics of BCL3 expression in BW5147 cells were studied by
RT-PCR after IL-9 stimulation for various periods of time (15 minutes
to 48 hours). As shown in Fig 2, the
BCL3 message is upregulated within 1 hour of IL-9 stimulation,
whereas maximal expression is reached upon 4 to 8 hours of stimulation.
View larger version (58K):
[in this window]
[in a new window]
| Fig 2.
Kinetics of BCL3 upregulation by IL-9. BW5147
cells were cultured in the absence or in the presence of 100 U/mL IL-9
for the indicated periods of time. Total RNA was extracted and RT-PCR
amplification was performed with oligonucleotides specific for
BCL3 or -actin. A Southern blot of the PCR products
was performed and filters were hybridized with the BCL3 or
-actin cDNA probes.
|
|
IL-9 induces delayed, IB-independent NF-B binding.
Because BCL3 has been reported to interact with NF-B
dimers,12,40 we studied NF-B DNA binding in
IL-9-stimulated BW5147 lymphoma. Cells were stimulated with IL-9 for
periods of time varying from 12 minutes to 24 hours. Nuclear extracts
were prepared and analyzed in a band shift assay with a
32P-labeled palindromic NF-B probe or a Sp1 probe as a
control. As shown in Fig 3A, at least 10 hours of IL-9 stimulation are required to observe a significant
increase in NF-B binding, and maximum levels of binding are reached
after 21 hours of stimulation. Using Sp1 binding as a control, NF-B
binding was quantified with a PhosphorImager and we found an average
fold induction of 2.8- ± 0.3-fold from 7 independent experiments
with 24 hours of IL-9 stimulation (P < .0001;
t-test). The plateau of NF-B binding was stable upon
long-term stimulation by IL-9 (at least 4 days; data not shown). By
Western blot analysis, the kinetics of upregulation of the BCL3 protein
correlated with NF-B DNA binding activity (Fig 3B). A similar
increase in NF-B binding in response to IL-9 was observed in other
cell lines in which IL-9 also upregulates BCL3 expression, such
as the TS2 T-helper clone and the MC9 mast cell line (data not shown).
View larger version (62K):
[in this window]
[in a new window]
| Fig 3.
IL-9 increases NF-B DNA binding. (A) EMSA analysis of
IL-9-stimulated BW5147 cells. Cells were cultured in the absence or in
the presence of 100 U/mL IL-9. Nuclear extracts were prepared after
various stimulation times and a mobility shift assay was performed
using a 32P-labeled palindromic B site. An Sp1 DNA
binding oligonucleotide was used as a control for the loading of
similar protein quantities in each lane. (B) Western blot analysis of
BCL3 induction by IL-9. The left panel shows BCL3 expression in BW5147
cells transfected with a control vector ( ) or the BCL3 cDNA
cloned into the pEF-BOS plasmid (BCL3) as a positive control. In the
right panel, cells were cultured as in (A). Total extracts were
prepared and analyzed by Western blot with anti-BCL3 antibodies as
described in Materials and Methods.
|
|
To compare the activity of IL-9 with that of a classical activator of
NF-B, BW5147 cells were stimulated for 5 minutes or 24 hours with
either IL-9 or TNF, and a band shift assay was performed with the
palindromic NF-B site. As shown in Fig
4A, 5 minutes of TNF stimulation was sufficient to result in a clear NF-B band shift, whereas IL-9-mediated activation is optimal only
after 24 hours. In addition to this difference in the kinetics of
activation, Fig 4A shows that NF-B complexes induced by TNF and IL-9
exhibited distinct eletrophoretic mobilities. IL-9-induced complexes
migrated faster than those induced by TNF. Moreover, antibodies against
the p65/RelA NF-B subunit supershifted TNF-induced complexes but not
IL-9-induced DNA binding (Fig 4B). By contrast, the latter complex is
supershifted by anti-p50. Because anti-p52 antibodies did not affect
IL-9-induced complexes (data not shown), the IL-9-induced
NF-B-binding complexes are likely composed of p50 homodimers.
View larger version (80K):
[in this window]
[in a new window]
| Fig 4.
Differences in NF-B binding induction by IL-9 and TNF.
(A) Delayed induction by IL-9 of a fast-migrating NF-B complex.
BW5147 cells were stimulated in the absence or in the presence of IL-9
(100 U/mL) or TNF (10 ng/mL) for 5 minutes or 24 hours. Nuclear
extracts were prepared and a mobility shift assay was performed using a
32P-labeled palindromic B site. The two types of NF-B
dimers binding to the probe (p65/p50 and p50 homodimers) are indicated.
(B) NF-B complexes induced by IL-9 contain only p50 subunits. BW5147
cells were stimulated in the absence or in the presence of IL-9 (100 U/mL) or TNF (10 ng/mL) for 24 hours. Nuclear extracts were prepared
and a mobility shift assay was performed using a
32P-labeled palindromic B site in the presence of
anti-p50 (4 µg per lane) or anti-p65 (2 µg per lane) antibodies.
The two types of NF-B dimers binding to the probe (p65/p50 and p50
homodimers) are indicated. (C) IL-9 does not induce the phosphorylation
and degradation of IB. BW5147 cells were stimulated with 100 U/mL
IL-9 or 10 ng/mL TNF for 5, 10, or 30 minutes or were left
unstimulated. Total cell extracts were loaded on a gel and Western
blotted. The membrane was hybridized with two antibodies: one directed
against total IB and another specific for phosphorylated IB
(Ser32).
|
|
All known inducers of NF-B cause phosphorylation-dependent
degradation of IB, which allows NF-B dimers to migrate freely into the nucleus.9 We analyzed the phosphorylation and
expression of IB by performing a Western blot with two different
antibodies: one recognizing total IB protein and one specific for
the Ser32-phosphorylated form of IB. As shown in Fig
4C, 5 minutes of TNF stimulation resulted in a transient IB
phosphorylation in BW5147 cells. The protein was then degraded and
began reappearing after 30 minutes of TNF stimulation. Unlike
TNF, IL-9 did not induce a phosphorylation/degradation of IB,
indicating that IL-9 does not trigger the classical IB-dependent pathway to activate NF-B.
Because the unusual kinetics of NF-B binding and the nature of the
dimers activated by IL-9 pointed to a role for BCL3 in this process, we
transfected the MC9 mast cell line with the BCL3 cDNA under the
control of the EF1 promoter to obtain a strong constitutive
expression of BCL3. When compared with control MC9 cells,
BCL3-expressing transfectants showed an increased nuclear NF-B activity that is similar to the effect of IL-9 on control cells. In addition, IL-9 failed to further increase NF-B binding in
BCL3-expressing transfectants (Fig
5). In 5 independent experiments, the IL-9-dependent NF-B induction
was quantified as a 2.9- ± 0.3-fold increase in control MC9 cells
(P = .0001; t-test), compared with a 1.1- ± 0.4-fold increase in BCL3 transfectants (P = .6; t-test).
View larger version (91K):
[in this window]
[in a new window]
| Fig 5.
Similar NF-B DNA binding induced by IL-9 and BCL3. MC9
mast cells transfected with either a control vector or the pEF-BOS
plasmid containing the BCL3 cDNA were stimulated with 100 U/mL
IL-9 for more than 24 hours or were left unstimulated. Shown is a
representative EMSA performed with the palindromic NF-B probe. An
Sp1 DNA binding oligonucleotide was used as a control for the loading
of similar protein quantities in each lane.
|
|
IL-9 inhibits TNF-induced NF-B-dependent transcription.
The role of BCL3 in NF-B-mediated transcription is still
controversial, because both stimulatory and inhibitory activities of
BCL3 have been reported.12,13,17,22,41,42 To analyze the
effect of IL-9 on NF-B transcriptional regulation, two palindromic NF-B binding sites were cloned upstream of the TK promoter followed by a luciferase reporter gene to obtain the pGL3-TK-BPD construct. As shown in Fig 6A, IL-9 did not
significantly affect the basal luciferase expression driven by this
promoter in BW5147 cells. However, 48 hours of IL-9 stimulation
drastically inhibited TNF-induced transcriptional activity (Fig 6A,
upper panel). Importantly, 8 hours after TNF stimulation, IL-9 could
only inhibit TNF-induced NF-B transcriptional activity when cells
were preincubated with IL-9, supporting the role of BCL3
induction in this process (Fig 6A, middle and lower panels). As
expected from the observation that IL-4 induces BCL3 expression
in BW5147 (Fig 1), this cytokine also partially inhibited the activity
of TNF under the same experimental conditions (Fig 6B). To directly
examine the effect of BCL3 on TNF-mediated NF-B activation, we
transiently transfected COS cells with the pGL3-TK-BPD reporter
plasmid and a BCL3 expression construct. As shown in
Fig 7, BCL3 expression completely inhibited TNF-induced luciferase activity.
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
IL-9 and IL-4 repress TNF-induced NF-B-dependent
transcription. (A) BW5147 cells were transfected with the
pGL3-TK-BPD plasmid and pRL-TK as an internal control and were
stimulated with IL-9 (100 U/mL) or TNF (10 ng/mL) or were left
unstimulated. In the upper panel, cells were cultured with or without
cytokines for 48 hours before luciferase assay. In the middle panel,
cells were stimulated only for 8 hours. In the bottom panel, cells were
incubated or not incubated with IL-9 for 48 hours, and TNF was added
for the last 8 hours before luciferase assay. The results are expressed
as arbitrary units of luciferase activity, with 1 U being defined as
the luciferase activity in unstimulated cells. (B) BW5147 cells were
transfected with pGL3-TK-BPD plasmid and pRL-TK as an internal
control and stimulated with IL-4 (500 U/mL) with or without TNF (10 ng/mL) or were left unstimulated. Luciferase was monitored after 48 hours.
|
|
View larger version (15K):
[in this window]
[in a new window]
| Fig 7.
BCL3 represses TNF-induced NF-B-dependent
transcription. COS cells were transiently transfected with
pGL3-TK-BPD, pRL-TK and either the BCL3 cDNA in the pEF-BOS
plasmid or the empty vector. After 24 hours, cells were stimulated or
not stimulated with TNF (10 ng/mL). Luciferase activities were
monitored 8 hours later. The results correspond to the mean ± SD of 4 individual transfections and are expressed as arbitrary units of
luciferase activity, with 1 U being defined as the luciferase activity
in unstimulated cells.
|
|
Role of STAT factors in BCL3 and NF-B induction by IL-9.
The increase in BCL3 mRNA induced by IL-9 could result either
from a transcriptional activation or from stabilization of the BCL3 message. To address this question, cells preincubated in the presence of IL-9 were washed and further cultured for up to 3 hours
with or without IL-9 and actinomycin D, an inhibitor of RNA synthesis.
After 3 hours, the BCL3 message was barely detectable without
IL-9, whereas a strong expression was maintained in the presence of
IL-9. By contrast, with actinomycin D, this effect of IL-9 was
completely abolished, indicating that transcription is required for
this activity (Fig 8). To determine whether
IL-9 directly upregulates BCL3 expression or whether this
process requires protein synthesis, BW5147 cells were stimulated with
IL-9 in the presence of cycloheximide. As shown in
Fig 9, cycloheximide did not affect
BCL3 expression, as assessed by RT-PCR analysis, indicating that new protein synthesis is not required for this IL-9 activity.
View larger version (32K):
[in this window]
[in a new window]
| Fig 8.
BCL3 upregulation by IL-9 is dependent on RNA
transcription. BW5147 cells were preincubated with 100 U/mL IL-9 for 24 hours, extensively washed, and restimulated for 1, 1.5, 2, 2.5, and 3 hours with or without IL-9 and actinomycin D (5 µg/mL) as indicated
in the figure. Total RNA was extracted and RT-PCR amplification was
performed with oligonucleotides specific for BCL3 or
-actin. A Southern blot of the PCR products was performed
and filters were hybridized with a BCL3 or -actin
oligonucleotide. (A) shows the autoradiography of the blot. (B) shows
the intensity of the BCL3 signals from the same blot quantified
by PhosphorImager analysis and standardized to the
-actin level.
|
|
View larger version (76K):
[in this window]
[in a new window]
| Fig 9.
New protein synthesis is not required for BCL3
upregulation by IL-9. BW5147 cells were stimulated with 100 U/mL IL-9
and with or without the combination of 10 µg/mL cycloheximide and 50 µmol/L MG-132 (a proteasome inhibitor) for 4.5 hours, or the cells
were left unstimulated. Total RNA was extracted and RT-PCR
amplification was performed with oligonucleotides specific for
BCL3 or -actin. A Southern blot of the PCR products
was performed and filters were hybridized with the BCL3 or
-actin cDNA probes.
|
|
To analyze further the mechanisms of BCL3 upregulation in
response to IL-9, we took advantage of BW5147 transfectant cells expressing mutated forms of the human IL-9 receptor (hIL-9R). When
cells were transfected with the wild-type hIL-9R, hIL-9 induced a
similar BCL3 expression to that induced by mIL-9 in the
parental cells (Fig 10). By contrast,
hIL-9 was inactive in cells expressing a mutated form of hIL-9R
(phe116) in which tyrosine residue 116 was replaced by a phenylalanine,
resulting in the inability of the receptor to activate STAT
transcription factors.31 To determine the respective role
of the different STAT proteins activated by IL-9 (STAT1, 3, and 5),
other hIL-9R mutants that specifically activate STAT5 (mut7) or STAT1
and 3 (mut6) were stably transfected in BW5147. As shown in Fig 10,
STAT5 is not necessary for BCL3 expression, because this gene
is still induced in BWmut6 cells (in which STAT5 is not activated in
response to hIL-9). In addition, STAT5 is not sufficient for
BCL3 expression, because this gene is not significantly
upregulated in BWmut7 (in which STAT5 is the only STAT activated by
hIL-9). As expected, the NF-B activation measured by EMSA in BW5147
transfected with the different hIL-9R mutants matched BCL3
expression (Fig 11). The mean NF-B DNA
binding induction in response to IL-9 were 3.9- ± 0.5-fold
(P = .01; t-test) for BWh9R, 1.3- ± 0.2-fold
(P = .19; t-test) for BWphe116, 5.1- ± 0.6-fold
(P = .016; t-test) for BWmut6, and 1.5- ± 0.2-fold (P = .26; t-test) for BWmut7.
View larger version (49K):
[in this window]
[in a new window]
| Fig 10.
BCL3 upregulation by IL-9 in cells expressing
mutated hIL-9R. BW5147 cells transfected with the wild-type human IL-9R
(BWh9R) or mutants of this receptor partially (BWmut6 and BWmut7) or
totally (BWphe116) defective in STAT activation (see text) were
stimulated with 500 U/mL human IL-9 for 24 hours. After electrophoresis
of 5 µg of total RNA and transfer to nitrocellulose, filters were
hybridized with a 32P-labeled mouse BCL3 cDNA
probe. Hybridization with a -actin probe confirmed the even
loading of RNA in each lane.
|
|
View larger version (36K):
[in this window]
[in a new window]
| Fig 11.
NF-B DNA binding induced by IL-9 in cells expressing
mutated hIL-9R. BW5147 cells transfected with the wild-type human IL-9R
(BWh9R) or mutants of this receptor partially (BWmut6 and BWmut7) or
totally (BWphe116) defective in STAT activation (see text) were
stimulated with 100 U/mL human IL-9 for 24 hours or with the equivalent
amount of mIL-9 as a positive control. Nuclear extracts were prepared
and a mobility shift assay was performed using a
32P-labeled palindromic B site. An Sp1 DNA binding site
was used as a control for the even loading of proteins in each lane.
|
|
| |
DISCUSSION |
Although BCL3 was cloned several years ago as a putative
proto-oncogene associated with human B-cell leukemias, its function in
the immune system began to be unravelled only recently, with the
analysis of knock-out mice.3,4 These mice display severe defects in antigen-specific immune responses, which clearly points to a
role for BCL3 in the regulation of the immune system. However, so far,
no clear picture of how BCL3 is regulated can be drawn up. In
this report, we identify IL-9 as the first physiological stimulus
triggering BCL3 expression in T cells and in mast cells. In
addition, we show that IL-4 can also induce BCL3 expression in
BW5147 lymphoma cells and T-helper cell clones.
The mechanisms underlying this induction were studied using IL-9R
mutants defective in STAT activation. STAT1 and STAT3 were found to be
particularly important for BCL3 expression in BW5147 stimulated
with IL-9. In line with this observation, STAT1 and STAT3 are also
activated by IL-4 in these cells (our unpublished observation). By contrast, interferon  activated only
STAT1 and did not induce BCL3 upregulation in BW5147 (data not
shown), suggesting that STAT3 is the most important (if not the only)
factor involved in BCL3 upregulation by IL-9 and IL-4. IL-6,
which activates STAT3 weakly in BW5147 (our unpublished
observation), induced a faint BCL3 expression
that is detectable by more sensitive techniques, such as RT-PCR (data
not shown). The fact that STAT proteins are transcription factors and
that BCL3 upregulation by IL-9 required RNA transcription but
not protein synthesis indicates that this process is directly regulated
at the transcriptional level.
As expected from several reports,17,18,43 BCL3
expression was followed by an increase in the DNA binding of p50
homodimers. The role of BCL3 in our system is supported by the
following observations: (1) anti-p50, but not anti-p65 antibodies
supershifted NF-B dimers that were activated by IL-9; (2) IL-9 did
not induce IB phosphorylation and degradation; (3) BCL3 protein
expression correlated with the late NF-B DNA binding induced by
IL-9; (4) cells transfected with the BCL3 cDNA showed a
constitutive DNA binding activity of p50 homodimers that is similar to
that induced by IL-9 and is not modified further by IL-9 stimulation;
and (5) in cells transfected with mutants of the IL-9R, we found a good
correlation between the level of BCL3 expression and the DNA
binding of p50 homodimers.
The function of BCL3 as a regulator of NF-B-dependent transcription
is still a matter of debate. Based on transient transfection experiments using NF-B and BCL3 expression plasmids, some
reports suggest that BCL3 promotes B-dependent
transcription,12,17 and others suggest that BCL3 inhibits
B-dependent transcription.22 We found here that IL-9
repressed TNF-dependent transcriptional activation through two
palindromic B sites, and a similar effect was observed with IL-4.
This repression requires preincubation in the presence of IL-9 for at
least 24 hours, in agreement with the kinetics of BCL3
induction. The role of BCL3 in this process is further supported by the
observation that BCL3 transient transfection similarly inhibited
TNF-dependent NF-B activity.
Interestingly, the IL-9 effect on NF-B-mediated transcription was
dependent on the nature of NF-B binding sites cloned in the reporter
construct. For example, no repression was observed with a reporter
plasmid containing NF-B sites from the TNF promoter (data not
shown). Such differential activity may result from the fact that p50
homodimers could more efficiently compete with p65/p50 heterodimers for
a palindromic sequence than for other types of NF-B sites. Taken
together, these observations suggest that IL-9 may specifically
downregulate a particular set of genes induced by NF-B. Further
experiments will have to identify these genes and to determine their
role in IL-9 activities in vivo.
In particular, the role of BCL3 in IL-9-induced tumors needs further
investigation. However, BCL3 is unlikely to play a role in the
antiapoptotic activity of IL-9, because BCL3 transfection does not
protect T-cell lymphomas against corticoid-induced apoptosis (data not
shown) and because this activity of IL-9 could be mediated by both
STAT5- and STAT3-activating mutants of the IL-9 receptor (Demoulin et
al, manuscript in preparation).
In conclusion, the data reported here shed some light on BCL3
regulation and point to a link between the Jak-STAT signal transduction pathway and the NF-B system. The identification of genes that are
regulated by cytokines such as IL-9 and IL-4 through this process could
lead to a better understanding of the mechanisms underlying the role of
BCL3 and these cytokines in immune regulation.
| |
ACKNOWLEDGMENT |
The authors thank Drs S. Nagata, A. Burgess, and C. Wildmann for their
generous donations of reagents and Dr L. van Fiets and V. Bours for
helpful discussions.
| |
FOOTNOTES |
Submitted September 11, 1998; accepted February 10, 1999.
Supported in part by the Belgian Federal Service for Scientific,
Technical and Cultural Affairs and the Opération
Télévie. M.R. and J.L. are scientific associates
(Télévie), J.-B.D. is a research assistant, and J.-C.R. is
a research ass |
TOP
ABSTRACT
INTRODUCTION