From the Laboratoire d'Immunologie Cellulaire, CNRS URA 625, Bat. CERVI, Hôpital de la Pitié Salpêtrière, 83, Bd. de l'Hôpital, 75013 Paris, France
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ABSTRACT |
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RelA and RelB belong to the nuclear factor-B
(NF-
B-Rel) transcription factor family. Both proteins are
structurally and functionally related, but their intracellular and
tissue distributions are different. In resting cells, RelB is found
mostly in the nucleus, whereas RelA is sequestered in the cytosol by
protein inhibitors, among which I
B
is the dominant form in
lymphocytes. Upon cellular activation I
B
is proteolyzed, allowing
RelA dimers to enter the nucleus and activate target genes. To study
the selectivity of gene regulation by RelA and RelB, we generated T
cell lines stably expressing a dominant negative mutant of I
B
. We
show that selective inhibition of RelA-NF-
B decreased induction of NFKB1, interleukin-2, and interleukin-2R
genes but not
c-myc. Transcription driven by the I
B
promoter was
blocked by the transgenic I
B
; however, wild type I
B
was
expressed in the transgenic cell clones but with much slower kinetics
than that in control cells. Wild type I
B
expression was
concomitant with RelB up-regulation, suggesting that RelB could be
involved in transcription of I
B
through binding to an alternative
site. These results indicate that RelB and RelA have both distinct and
overlapping effects on gene expression.
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INTRODUCTION |
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Nuclear factor B
(NF-
B)1 is part of the Rel
family of eukaryotic transcription factors which share structural and
functional properties. Although ubiquitously expressed in higher
eukaryots NF-
B has been intensively studied mostly in cells
belonging to the immune system where it was first discovered (for
review, see Refs. 1 and 2). NF-
B-Rel factors were shown to
participate in the expression of genes essential for the immune
responses and to regulate gene transcription during inflammatory
reactions. The prototypical NF-
B is a homodimer or heterodimer
composed of 50-kDa (p50) and/or 65-kDa (p65 or RelA) polypeptides. In
vertebrates other members of the family are c-Rel, RelB, and p52. The
tissue and cellular distribution of the three last members is more
restrained than that of the prototypical NF-
B. For example, the
expression of RelB was described as being predominant in dendritic
cells from primary and secondary lymphoid organs (3-6). RelB has also been detected in other cells and tissue but in lower amounts or after
specific activation. c-Rel and p52 are also expressed mainly in cells
from the hematopoietic lineages. P50 and p52 are generated by
proteolytic processing of precursor polypeptides (p105
(NFKB1 gene) and p100 (NFKB2 gene), respectively)
(1, 7). Each member of the NF-
B-Rel family contains a 300-amino acid
sequence called the Rel homology domain, which is critical for nuclear translocation, protein-protein interactions, and sequence-specific DNA
binding. All members of the NF-
B-Rel family form dimers. The dimers
can be classified into two pools on the basis of their intracellular
localization, which is critical in regulating their activity. One pool
of NF-
B-Rel dimers is cytosolic in the absence of cellular
activators, whereas the second pool is constitutively nuclear. The
intracellular location of the dimers depends on the capacity of the
NF-
B-Rel family members to interact with ankyrin repeat-containing
proteins, collectively called I
B. The cytoplasmic I
Bs inhibit
NF-
B-Rel complexes by preventing both NF-
B-Rel nuclear
translocation, and their interaction with specific decameric DNA
sequences called
B (8, 9). Thus I
Bs represent intracellular regulators of NF-
B activity. Several members of I
B regulatory family have been characterized, including I
B
, I
B
, I
B
,
the two NF-
B protein precursors p105 and p100, and bcl-3. Except for
bcl-3, I
B molecules are mostly cytosolic, although nuclear I
B
has been reported in cultured cells (10-12) and in
vivo.2 p50 and p52
homodimers as well as RelB-p50 and RelB-p52 heterodimers do not
interact efficiently with cytosolic I
Bs. Consequently they are found
in nuclei of cells that produce these complexes (13). Therefore their
regulation should be distinct from the cytosolic forms of NF-
B. The
p50 and p52 homodimers were reported to interact with the nuclear
bcl-3. The resulting trimers seem to constitute transcriptional
activators, whereas p50 and p52 homodimers are unable to enhance RNA
polymerase II-driven transcription (14). In contrast to other members
of the NF-
B family, RelB contains in its NH2-terminal
domain a leucine zipper-like structure that is essential for
transactivation of target genes (15). However, the regulation of RelB
activity is still poorly understood.
Studies of T lymphocytes, isolated from IB
-deficient mice,
demonstrated that the dominant I
B regulator of NF-
B-Rel is I
B
, the product of the MAD3 gene (16, 17). Activation
of cells with adequate signals such as T cell receptor triggering, phorbol esters, interleukin 1 (IL-1), tumor necrosis factor (TNF-
), and others results in I
B
degradation by 26 S proteasomes (for review, see Ref. 7). This renders dimers, which contain RelA and c-Rel
proteins, free to translocate into nuclei where they activate
transcription of target genes. The molecular mechanism resulting in
I
B
proteolysis is complex and not completely elucidated. However,
at least two post-translational covalent modifications have been
reported to be essential for its degradation. The first critical event
is phosphorylation of serines 32 and 36 in the NH2-terminal
region of I
B
, carried out by Ser/Thr kinase(s) including a
multienzyme complex of 700 kDa (18). This double phosphorylation of
I
B
does not lead to dissociation from NF-
B, but it is
prerequisite for the second modification step, which is the
ubiquitination of two NH2-terminal lysines at positions 21 and 22 (19). Subsequently the phosphorylated and ubiquitinated I
B
is proteolyzed by the 26 S-proteasome complex (20, 21).
Once released from IB
, NF-
B-Rel proteins translocate rapidly
to the nucleus where they exert their regulatory functions by
interacting with specific decameric
B sequences and the general transcription factor TFIIB (22). A plethora of genes have been shown to
contain
B sequences in their promoters (for review, see Ref. 23). In
T cells, gene products involved in cell adhesion (intercellular cell
adhesion molecule-1; ICAM-1), cell growth control (IL-2, its receptor
IL-2R
, and c-myc), and proinflammatory mediators (IL-6, TNF-
) are
suspected of being transcriptionally regulated by NF-
B. Furthermore,
viruses with T cell tropism, such as HIV, are also thought to be
transcriptionally regulated by NF-
B proteins (24). Specific
relationships between distinct NF-
B complexes and particular target
genes are not yet understood, although preferential binding and
preferential transcriptional activation efficiencies have been
demonstrated by transfection experiments with discrete NF-
B
expression vectors and distinct
B sequences (25-29). These
observations suggest that distinct NF-
B-Rel complexes modulate
transcription of different genes selectively.
Transient transfection assays with a mutated form of IB
, in which
serines 32 and 36 were replaced by alanines, demonstrated that the
double mutation prevented proteolytic degradation of the transgenic
I
B
by the usual NF-
B activators (TNF-
and phorbol esters)
(30-32). Thus the double mutation generates a constitutive repressor
of the cytosolic NF-
B-Rel proteins. Because the 32/36A I
B
mutant (I
B
32/36A) should not affect the
constitutively nuclear pool of RelB proteins,
I
B
32/36A potentially represents a powerful and
selective tool for the study of the respective roles of NF-
B and
RelB protein complexes in gene expression. We have therefore used cell
clones that express both RelA and RelB subunits for stable
transfections with the I
B
32/36A. We report in the
present publication the effects of the expression of the transgenic
I
B
on NF-
B-Rel activation and gene expression.
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MATERIALS AND METHODS |
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Cells-- The parental HPB-ALL cell line was cultured in RPMI 1640 medium containing glutamine, antibiotics, and 10% fetal calf serum.
Stable Transfections--
Expression vector pCMV-
IB
32/36A was made by inserting MAD3
cDNA mutant at amino acid positions 32 and 36 into the
XbaI/HindIII sites of the pcDNA3 vector from
Invitrogen (S32/36A mutant in 32). HPB-ALL cells were transfected with
pCMV-I
B
32/36A and the empty pcDNA3 vector by
electroporation. G418-resistant cells were cloned by limiting dilution
and genotyped by Southern blotting (33), using a full-length
MAD3 cDNA probe. The clones with stable integration of
I
B
32/36A were grown in RPMI with 10% fetal calf serum in presence of 1 mg/ml G418.
Cell Extracts-- In gel shift experiments (electrophoretic mobility shift assay; EMSA) cells were incubated for the indicated periods of time with activators, and nuclear proteins were extracted as described previously (34). In Western blotting, the cytosolic extracts were obtained in the hypotonic buffer described in Ref. 34.
Western Blotting--
Equal amounts of protein (30 µg)
extracted from cytoplasma of control or
IB
32/36A-transfected clones were fractionated on 10%
polyacrylamide gels by electrophoresis in denaturing conditions, according to Porzio and Pearson (35). Proteins were electrotransferred onto polyvinylidene difluoride membranes (Millipore). The efficacy of
the transfer was tested by Ponceau Red staining. The wild type and
transgenic I
B
were determined using a monoclonal antibody (MAD
10B) specific for an NH2-terminal domain of I
B
(36). The antigen-antibody complex was revealed using horseradish
peroxidase-coupled anti-mouse antibody and the Amersham enhanced
chemiluminescence visualization system (ECL) kit. The autoradiography
was carried out for 5 s to 10 min. For RelB Western blotting, the
identical procedure was followed except that 70 µg of nuclear
extracts was used. The RelB-specific antiserum was from Santa Cruz
(Tebu, France), and its dilution was 1/500.
EMSA--
The EMSA was performed using 10 µg of nuclear
protein extracts/incubation. The B oligonucleotide used was a kind
gift from Dr. Leo Lee (NCI, Frederick, MD) and corresponds to the
tandem
B sequence (PRE) from the HIV LTR. The gel shift experiments were carried out following the procedure described in Ref. 34. To
identify the PRE-binding proteins, nuclear extracts from control HPB-ALL cells and one of the stably transfected clones, the A3 clone,
were incubated with 2 µl of antibodies specific for individual NF-
B-Rel proteins before the addition of the radiolabeled PRE oligonucleotide. All antibodies were purchased from Santa Cruz. In
addition to the Santa Cruz antibodies, we also used an antiserum specific for the COOH-terminal domain of the RelA molecule (named 1226 in Ref. 37), kindly provided by Dr. Nancy Rice (NCI).
Chloramphenicol Acetyltransferase (CAT) and Luciferase (Luc)
Assays--
The following vectors were used for
B-dependent CAT and Luc assays. The 1168hIL6Luc+
construct, which contains 534 base pairs of the human IL-6 promoter,
was kindly provided by Prof. G. Haegeman (Gent University, Belgium).
Dr. A. Israël (Pasteur Institute, France) provided us with the
1.2HN-Luc construct (38) containing the NFKB1 (p105)
promoter region and the 0.4SK, 0.2SK, and 0.4SK68
B Luc plasmid
containing the MAD3 (I
B
) promoter constructs (39). The
0.4SK contains all three
B sites from the MAD3 promoter
domain, whereas the 0.2SK contains only the proximal
B1 site, and
the 0.4SK
B contains only the
B2 and
B3 sites. To monitor
the tranfection of the three constructs of MAD3 promoter,
the p.
gal-promoter vector (CLONTECH), which
contains a functional LacZ gene downstream of the SV40 early
promoter, was cotransfected, and the
-galactosidase activity was
measured by spectrophotometry in the presence of 100 nM
o-nitrophenol
-D-galactoside. The ICAM-1
promoter-Luc construct (pGL1.3) was described by Ledebur and Parks (40)
and was provided by Dr. K. Roebuck (Rush, Chicago). The
c-myc promoter (
2325 to +36) and c-fos promoter
(
711 to +42) CAT constructs are described in Ref. 41. The LTR3
CAT-218 construct containing the 218 base pairs upstream from the
transcription initiation start of the HIV 5
-LTR (42) was provided by
Dr. R. B. Gaynor (UCLA, Los Angeles). Finally, Dr. G. R. Crabtree
(HHMI, Stanford, CA) provided us with the IL-2 promoter-Luc construct
(pCLN15
CX) (
326 to +45). Transient transfections of the cell
clones with CAT and Luc vectors were performed by electroporation at
200 V, 500 microfarads (Bio-Rad electroporation system) with 20 µg of plasmid DNA/5·106 cells. 2 h after transfection,
cells were split into two pools. One pool of cells was incubated in
RPMI (untreated cells), and the other pool was incubated with 10 ng/ml
phorbol 12-myristate 13-acetate (PMA) plus 1 µg/ml phytohemagglutinin
(PHA) for 24 h (activated cells). The cells were then collected,
washed once with phosphate-buffered saline, and lysed by three cycles
of freezing/thawing in 150 mM Tris-HCl, pH 8. Cell
extracts, normalized for total protein content (43), were assayed for
CAT activity using [14C]chloramphenicol (NEN Life Science
Products) according to Gorman et al. (44). The
chloramphenicol conversion was quantified using a BetaImager 1200 apparatus (Biospace, France). The results were expressed as percent of
chloramphenicol conversion/mg of protein (relative CAT units).
Transfection experiments were repeated at least three times, using two
independent plasmid preparations. Luc assays were performed using the
Promega luciferase assay system. The cells were lysed with 25 mM Tris phosphate, 2 mM dithiothreitol, 2 mM
1,2-diaminocyclohexane-N,N,N
,N
-tetraacetic
acid, 10% glycerol, 1% Triton X-100, pH 7.8. The light emission was
measured in a luminometer (Bio-Rad). The results were calculated as
relative light units (light emission/background/mg of protein).
Northern Blotting--
cDNA probes used for Northern
blotting were obtained by enzymatic digestion of the following vectors:
pKH47-c-myc (PstI/EcoRI digestion generating a
1,200-base pair fragment of the c-myc cDNA), p1IL-2
(PstI/BglII digestion generating a full-length
cDNA), pBRchIL6F2 (PstI digestion generating an 855-base
pair fragment of the human IL-6 cDNA), p105
(HindIII/ApoI digestion generating full-length cDNA), pCMV-MAD3 (HindIII/XbaI digestion
generating full-length IB
cDNA), pBr322-actin
(PstI digestion generating full-length actin cDNA). The
cDNA probes were radiolabeled using [
-32P]dCTP
(Amersham) and the Rediprime kit (Amersham). Total cytoplasmic RNA was
prepared according to a modified method of Chomczynski and Sacchi (45),
using the Stratagene RNA kit. Total RNA (10-20 µg) was fractionated
by electrophoresis on 0.7% agarose gels containing 2.2 M
formaldehyde. Gels were blotted on Hybond N+ membranes
(Amersham) according to the indications of the manufacturer. Membranes
were hybridized with 32P-labeled probes in Quickhyb
solution (Stratagene) according to the protocol supplied by the
manufacturer, at 65 °C. Membranes were autoradiographed for 1-12 h
at
70 °C with intensifying screens. Membranes were stripped by
boiling in H2O and rehybridized with the
-actin probe to
normalize loading of RNA samples.
Measurement of IL-2 Production by Enzyme-linked Immunosorbent Assay (ELISA)-- Control, A3, and D7 clone cells (105 cells/condition) were activated by PMA (10 ng/ml) plus PHA (1 µg/ml) for 12 h at 37 °C. Cell supernatants were tested for IL-2 by ELISA using the Immunotech human IL-2 ELISA kit (Immunotech, France). All assays were performed in quadruplicate.
Measurement of CD25 Expression by Flow Cytometry-- Control, A3, F10, and D7 cells were activated for 24 h by PMA (10 ng/ml) and PHA (1 µg/ml). Unstimulated cells and PMA plus PHA-treated cells were tested for CD25 by flow cytometry using a phycoerythrin-conjugated human CD25-specific monoclonal antibody from Caltag Laboratories and fluorescence-activated cell sorter apparatus from Becton-Dickinson.
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RESULTS |
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Characterization of Stable HPB-ALL Clones Transfected with the
32/36A Mutant IB
--
The parental HPB-ALL cell line is a T cell
tumor producing IL-2 and IL-6 in response to T cell activators, such as
phorbol esters, in the presence of a Ca2+ influx activators
(PHA, ionomycin, CD3-specific antibodies, etc.). Its phenotype is close
to a double positive thymocyte (CD4+/CD8+,
CD1a+, CD3+). We chose this line as a model for
studying the inhibition of the inducible NF-
B by a dominant negative
form of I
B
(I
B
32/36A). The stability of the
integration of the mutant I
B
was verified by Southern blotting of
DNA extracted from several clones isolated by limiting dilution and
cultured for 1 month in the presence of the selective antibiotic (not
shown). Three clones, A3, D7, and F10, were identified as stably
transfected with I
B
32/36A. To verify that the
I
B
cDNA was expressed in these clones, we performed Western
blot analysis of the cytosolic fractions of the control and the
"mutant" clones, using a monoclonal antibody specific for the
NH2-terminal domain of I
B
(36). The wild type and the
mutant I
B
are distinguishable on the basis of their electrophoretic migration because the 32/36A mutant migrates slightly slower in SDS gels (32). In the three clones that integrated I
B
32/36A, a slower migrating protein was specifically
detected by the antibody in addition to the wild type I
B
(Fig.
1A). Judging by the immunoblot
results, the mutant and wild type I
B
were expressed at comparable
levels in clone A3 and F10, whereas in clone D7, the mutant I
B
was more highly expressed relative to the wild type I
B
. In
control cells, only the faster migrating 36-kDa I
B
was detected.
In none of the mutant clones did expression of the transgenic I
B
prevent constitutive production of the wild type I
B
.
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IB
32/36A Blocks Nuclear Translocation of
RelA-NF-
B but Not of RelB-p50--
To investigate the duration of
NF-
B inhibition by the mutant I
B
, we analyzed NF-
B nuclear
translocation during a time course of PMA plus PHA treatment. In the
control cells,
B binding activities were clearly detectable at the
3 h time point and increased in intensity up to 24 h of
treatment (Fig. 2A). In the A3
clone, no significant
B binding activity was detected until 7 h
of activation. However, by the 7 h time point, a
B binding
activity, migrating as a doublet, was clearly detected and reached
levels similar to the control by 24 h of activation (Fig.
2A). To identify the proteins in the complexes bound to
B
oligonucleotide, we tested the abilities of antibodies specific for
RelA, c-Rel, p50, and RelB to affect the EMSA patterns of control and
A3 cells after 7 and 24 h of PMA plus PHA stimulation. In the
absence of specific antibodies, several complexes were detectable in
the control cells. The discrimination of these complexes was difficult
in this type of gels; but clearly, in the A3 clone, only two bands were
detectable after 7 h of cell stimulation, whereas in control cells
additional, slower migrating bands, existed (see Fig. 2B and
photographically enlarged view in Fig. 2C). Antibodies
specific for RelA and RelB demonstrated that the two upper bands from
the control cells contained RelA, whereas one of the lower bands
contained RelB (Fig. 2B). p50-specific antibodies removed
the two lower bands from both A3 and control cells. Thus, the upper
band in A3 clone was composed of RelB-p50 dimers, whereas the lower
band was the p50 homodimer. Antibodies specific for c-Rel had no effect
on the
B-binding proteins in control or A3 cell nuclear extracts
(Fig. 2), whereas they inhibited efficiently c-Rel-p50 binding in
control cells (not shown). Thus, whereas in control cells both RelA and
RelB dimers were detected, in the A3 clone only RelB-p50 and p50 dimers were detected. After 24 h of PMA plus PHA activation, both control and A3 nuclear extracts contained only the two faster migrating complexes (p50-p50 and RelB-p50) (Fig. 2B). These results
demonstrated that in control HPB-ALL cells, the initial effect of PMA
plus PHA activation led to nuclear translocation of cytosolic NF-
B proteins (RelA homo- and heterodimers). As expected, in the
I
B
32/36A-transfected A3 clone, the translocation of
these proteins was inhibited. Prolonged stimulation led to RelB
activation in both cell clones. Furthermore, after 24 h of PMA
plus PHA treatment activation of the RelA-containing complexes was also
inhibited. It was not surprising to observe comparable levels of RelB
in both control and A3 cell clones since RelB activation was reported
to be independent of I
B
. Similar results were obtained with the
two other I
B
32/36A-transfected F10 and D7 cell clones
(not shown). Thus, we have generated a cell system in which the
prototypical NF-
B is inhibited selectively by
I
B
32/36A, but activation of RelB remains potentially
intact. Western blotting analysis of nuclear extracts from A3 and
HPB-ALL cells further assessed the presence of RelB. RelB nuclear
amounts were increased upon PMA plus PHA stimulation (Fig.
3). In addition, immunochemical analysis
with RelB-specific antibodies confirmed the increase of RelB in nuclei
of control cells and A3 cells after 24 h of stimulation (not
shown). This is suggestive of a transcriptional, or at least,
pretranslational, regulation of the RelB in PMA plus PHA-activated
HPB-ALL T cells.
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IB
32/36A Expression Is Increased in
PMA-stimulated Cells--
In the absence of stimulation, wild type
I
B
has a rapid turnover that is independent of serines 32 and 36 phosphorylation and of ubiquitination (46). After stimulation with PMA
or TNF-
, I
B
is modified by phosphorylation and ubiquitination,
and the balance between degraded and newly synthesized I
B
turns
transiently in favor of the degradation (47). As a result, I
B
is
detected in lower amounts in cytosol from short term activated cells.
However, the resulting activation of NF-
B induces newly synthesized
I
B
that is detectable within 1-2 h after activation. This
neosynthesized I
B
is, in turn, probably responsible for the
inhibition of the RelA-containing NF-
B at later time points of PMA
plus PHA treatment (see Fig. 2B). This cycle of
activation-induced proteolysis/resynthesis of I
B
is initiated by
the phosphorylation of serines 32 and 36. To examine the fate of the
I
B
32/36A versus the wild type I
B
in
activated cells, we performed kinetic experiments in which the A3 and
the control clones were treated with PMA plus PHA for increasing
lengths of time. Western blot analysis of I
B
after up to 2 h
(Fig. 4A) and 24 h (Fig.
4B) of activation by PMA plus PHA showed that the wild type
I
B
was degraded almost completely within 30 min in control cells.
After 1 h of PMA plus PHA treatment it was resynthesized
progressively, reaching initial levels after as soon as 3 h of
activation (Fig. 4B). In the A3 clone, the wild type
I
B
was also degraded rapidly in response to cell activation, but
neosynthesized wild type I
B
was detectable only after 7 h of
PMA plus PHA treatment, reaching initial levels at the 9 h time
point (Fig. 4, A and B). In contrast to the wild
type I
B
, the I
B
32/36A was not degraded in
response to cell activation. In fact, levels of
I
B
32/36A increased from the 30 min time point to
reach a steady maximum at 2 h (Fig. 4A) probably
because the CMV promoter is activated independently of NF-
B
activation. These experiments clearly demonstrated the stability of the
mutant I
B
in activation conditions that lead to wild type
I
B
proteolysis.
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Differential Effect of IB
32/36A on Several
B-dependent Promoters--
To test the functional
consequences of the selective inhibition of RelA containing NF-
B, we
performed transient transfections of A3, D7, F10, and control cells,
with a series of reporter gene constructs (CAT or Luc) linked to
promoter regulatory regions of seven genes suspected to constitute
targets for NF-
B. In each of these promoters, at least one
B
consensus sequence was identified in addition to sites specific for
other regulatory transcription factors. The specificity of the
I
B
32/36A inhibition on NF-
B-driven transcription
was assessed with a reporter plasmid dependent on serum response
element (c-fos CAT) (41). The results are summarized in
Tables I and
II. All of the promoters used in this
study were activated by PMA plus PHA in control cells. The inductions
of CAT and Luc constructs by PMA plus PHA ranged from 2.2-fold
(c-myc) to 189-fold (p105) in control cells (Table I). The
effect of the I
B
32/36A transgene on activation of CAT
and Luc transcription depended on the promoter used. The transcription
of the reporter genes driven by HIV LTR, MAD3, IL-6, IL-2,
and p105 promoters was strongly inhibited in the three clones used
(Table II). For example, in clone A3, activation of the IL-2 promoter
reached only 3% of that obtained in control cells. In contrast, only
50% inhibition was observed with the ICAM-1 promoter. Finally,
c-myc promoter driven transcription of CAT was not inhibited
at all in the I
B
32/36A-transfected clones. Transient
transfections performed with the control fos-CAT showed no
difference in transcriptional activation between control cells and the
A3 clone and a small inhibition in the D7 and F10 clones. Together
these results indicated a selective effect of the I
B transgene on
NF-
B-driven transcription. Three sets of promoters could be
distinguished on the basis of their responsiveness to NF-
B
inhibition: promoters that were strongly inhibited by the transgene
(IL-6 and IL-2, for example), promoters that were partially inhibited
(ICAM-1), and promoters that were not affected by the lack of NF-
B
translocation (c-myc). Activation of the I
B
(0.2SK)
promoter, which was shown to contain the
B1 sequence responsible for
the transcriptional induction by RelA-p50 in Jurkat cells (39), was
abolished totally in two of the three clones (A3 and D7). To estimate
the potential contribution to I
B
-promoter activation by RelB
through binding to the two upstream
B sites, we performed Luc assays
with two additional I
B
-promoter constructs, the 0.4SK-Luc,
containing all three
B sites, and the 0.4SK
B, which contains
only the
B2 and
B3 upstream sites. With the 0.4SK-Luc construct,
only a little activation was obtained in A3 clone (2.1-fold) after PMA
plus PHA treatment compared with control cells (23.6-fold) (Fig.
5). The 0.4SK
B construct was not
activated by PMA plus PHA in control and A3 clones (Fig. 5), indicating
that
B2 and
B3 sites are not capable of enhancing I
B
transcription in the absence of the
B1 site. Together, these results
suggested that if RelB is implicated in I
B
gene up-regulation, it
does so through the involvement of a site(s) distinct from those
contained in the promoter region reported up to now. Thus, to determine
the impact of NF-
B inhibition on gene expression in the context of
the genome, we analyzed the mRNA and/or protein production of
several NF-
B target genes.
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|
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Effect of IB
32/36A on mRNA
Production--
We compared the induction of five
B target genes by
a time course analysis of mRNA production by Northern blotting.
Hybridization of the
-actin probe was used as control (Fig.
6). In the empty vector-transfected cell
clone (control), p105 (NFKB1), IL-2, wild type I
B
mRNA, and IL-6 (data not shown) were induced in a time course-dependent manner. In contrast, no transcripts for
IL-2 and IL-6 (data not shown) were detected in the A3 clone. The
NFKB1 (p105) transcript was produced constitutively in A3
cells, but no induction by PMA plus PHA was observed. In the control
cells, MAD3 (I
B
) mRNA reached a steady state after
as little as 30 min of PMA plus PHA treatment. In A3 cells, two
transcripts were detected with the MAD3 probe. The higher
mobility transcript, corresponding to the mutant I
B
, was strongly
augmented in the early time points of activation, peaking at 1 h,
whereas the slower migrating wild type messenger was detected at later
time points (6-24 h). Thus, whereas transcription of IL-2 and IL-6
genes was inhibited completely in the A3 clone, induction of the wild
type I
B
messenger RNA was delayed in the A3 clone compared with
the control cells. Finally, the c-myc messenger was produced
constitutively in these cells, and no induction by PMA plus PHA was
detected in either control or A3 cells.
|
Both IL-2 and IL-2R Productions Are Inhibited by the
I
B
32/36A Mutant--
We further investigated the
regulation of IL-2, an important T cell proliferation regulator, by
measuring its production at the protein level in two of the stable
clones (A3 and D7) by ELISA (Fig. 7). In
both transgenic clones, IL-2 production was 10% of the control after
12 h of PMA and PHA activation. Thus, the result obtained with the
Northern blot analysis of IL-2 induction was confirmed at the protein
level.
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DISCUSSION |
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In the present paper, we have reported the effect of a selective
inhibition of the RelA-containing NF-B on gene expression in T cell
clones. We have generated this cell system by stable transfection of a
mutant form of I
B
which has been shown previously to block
inducible RelA and c-Rel nuclear translocation (32). The particular T
cell line that we used also naturally expresses RelB. Three independent
cell clones, A3, D7, and F10, were selected by limiting dilution. In
agreement with previous reports (30-32), no proteolysis or
ubiquitination of the mutant I
B
was detected under conditions in
which signal-induced degradation of the wild type I
B
occurred. In
all three of the clones, the stability of the transgene led to an
efficient inhibition of the RelA-containing NF-
B DNA binding,
whereas RelB-p50 DNA binding capacity was unchanged, as expected.
Therefore the signal-activable NF-
B was inhibited selectively,
whereas the inducible I
B
-independent RelB-p50 complex remained
potentially active.
Expression of RelB is unusual in T cells. RelB is dominantly expressed
in dendritic cells from both primary and secondary lymphoid organs (3,
5, 6) and in B cells at later stages of development. Its involvement in
dendritic cells development was demonstrated clearly in RelB knockout
mice (4, 50), but the role of RelB in gene expression remains obscure.
Thus, our cell system is a convenient model for discriminating between
gene transcription regulated selectively by the RelA-NF-B and genes that may be regulated by RelB. In this respect, among the genes that we
have studied the regulation of I
B
expression is of particular interest. It has been shown, indeed, that among the three
B
consensus sites found in the human MAD3 promoter region, it
is the most proximal site,
B1, that mediates PMA and TNF activation
through binding of RelA complexes (39). The
B2 site is recognized by RelA complexes, but it is not able to mediate efficient activation of
the MAD3 promoter in cells producing only RelA and c-Rel
complexes. The
B3 site was unable to bind NF-
B proteins extracted
from myeloid cells (39). In HeLa cells all three
B sites were
reported to contribute efficiently to TNF-
activation of I
B
promoter (51). In addition, transfection of Jurkat cells with RelB
vectors led to increased levels of I
B
, suggesting that RelB is
capable of enhancing I
B
expression (13). We used three-luciferase reporter constructs that contain, respectively, all three (0.4SK), the
two upstream (0.4SK
B), or only the
B1 (0.2SK) sites of the
I
B
promoter. With these constructs the induced transcription of
Luc was inhibited potently in all of the I
B
32/36A
transgenic clones. This result demonstrates that in the MAD3
promoter, the three
B sites mediate I
B
transcription by
selectively binding forms of NF-
B which are themselves regulated by
I
B
. RelB was unable to compensate for the lack of NF-
B
activation in this assay. However, expression of I
B
did occur but
at later time points of PMA stimulation than in control cells. The
expression of the wild type I
B
mirrored the increase of RelB DNA
binding activity and protein. In addition, resynthesis of I
B
did
not occur in the MCF7 cells stably transfected with
I
B
32/36A, cells that do not express RelB (52). These
results strongly suggest that not only does RelB regulate I
B
transcription through a site different from that which binds the
prototypical NF-
B, but that NF-
B is not required for full
I
B
expression when RelB is produced in sufficient amounts. This
effect might be specific for human I
B
since the porcine I
B
promoter domain, which contains six
B consensus sites, was not
activated by overexpression of RelB (53). The alternative RelB-specific
regulation of I
B
could have functional consequences in cells that
produce high levels of RelB such as dendritic cells. In such cells,
NF-
B activity could be regulated negatively by I
B
overexpression due to to RelB. However, the correlation between
MAD3 expression and RelB activation remains to be
established.
The potential compensatory effect of RelB was not observed with all of
the genes that we examined. For example, expression of IL-2 was
inhibited dramatically at both RNA and protein levels. IL-2 promoters
possess multiple regulatory sequences among which are a single B
consensus site and multiple sites capable of interacting with c-jun
protein complexes. Expression of a dominant negative mutant of c-jun
abolishes IL-2 expression (54) probably because it coordinately blocks
the IL-2 transcriptional regulation at multiple sites. In activated T
cells, the major forms of NF-
B which bind to the
B site in the
IL-2 promoter were shown to be p50 homodimers (55) and RelA homo- and
heterodimers (56). Paradoxically, disruption of the c-rel
gene in mice also inhibited induced IL-2 production despite the
presence of RelA and p50 (57). A possible explanation of this effect
was reported recently by Smith Shapiro et al. (58), who show
that c-Rel regulation of the IL-2 promoter might be mediated by AP1
rather than directly through binding to
B sites. In contrast to
c-Rel, RelA was not able to activate AP1-dependent
luciferase expression (58). Here we show that in the absence of RelA
dimers, RelB-p50 cannot rescue IL-2 expression. Further, the degree of
IL-2 inhibition by I
B
32/36A transfection brings
additional strong evidence that activation of RelA dimers is a limiting
step for IL-2 transcriptional initiation.
Contrary to c-Rel disruption, inhibition of RelA dimers also diminished
expression of the IL-2 receptor (CD25). Therefore, the classical
NF-B dimers seem to be involved in regulating the whole IL-2 growth
control system.
In contrast to MAD3, RelB was not able to enhance the signal
induced expression of NFKB1 (p105), indicating that
selective activation of RelA dimers is required for the signal-induced
expression of p105. However, the p50 protein (the processed, functional
product of the NFKB1 gene) and the p105 mRNA were
produced in both parental and the IB
transgenic cells,
independent of cell activation. This suggests that the constitutive
expression of NFKB1 is independent of
B enhancers.
Interestingly, c-myc expression was not inhibited by the
inhibition of NF-B. The c-myc promoter upstream
B site
was shown to bind to RelA and c-Rel dimers and to be a positive
regulator of the c-myc promoter in CAT assays in B lymphoma
cells (59). Here we show that c-myc is expressed
constitutively, not only in the parental HPB-ALL cells, but also in the
I
B
32/36A transgenic cell clones. The c-myc
promoter activity was only feebly enhanced by PMA plus PHA, and it was
not decreased by the inhibition of RelA-NF-
B. It is therefore
possible that RelB is able to activate c-myc expression
constitutively. Alternatively, c-myc expression could be
independent of the
B sites in HPB-ALL cells.
HIV LTR contains two direct repeats of the B site in tandem. These
B sites are critical for the initial steps of HIV replication (24,
60, 61). It has been shown that although RelA-p50 up-regulates the HIV
promoter through binding to the
B tandem sequence, c-rel behaves as a repressor of the RelA-p50 in the context of HIV LTR and
the CD25 promoter (62). In control HPB-ALL cells, PMA plus PHA
activated the transcription from the HIV LTR by 42-fold over the basal
level. This activation was inhibited by 90% in the
I
B
32/36A transgenic cell clones. Thus RelB was unable
to substitute for RelA dimers. I
B
32/36A could be a
powerful tool for repressing HIV replication in infected cells.
However, since HIV replication is independent of NF-
B in the
presence of the HIV Tat regulatory factor (61), we are currently
investigating by infection experiments whether the effect seen in the
CAT assay can be extrapolated to the viral replicative cycle.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Alain Israël
and Douglas Ferris for reading the manuscript and for helpful
discussion and to Dr. Catherine Dargemont and Catherine Amarger for
helping with the graphical processing of the data. We are indebted to
Dr. Fernando Arenzana-Seisdedos for the IB
-specific monoclonal
antibody and to Dr. Patrick Baeuerle for providing the
I
B
32/36A expression vector. We thank Drs. A. Israël, R. B. Gaynor, C. G. Crabtree, K. Roebuck, A. Harel-Bellan, J. Wietzerbin, and G. Haegeman for providing the various
CAT and Luc constructs as well as some of the cDNA constructs used
in the Northern blot assays, and we especially thank Prof. Patrice
Debré for constant encouragement.
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FOOTNOTES |
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* This work was supported by the European Community Grant BiotechII BIO2-CT92-0130 and by a grant from the Agence Nationale de Recherche sur le Sida.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.: 33-1-4217-7513;
Fax: 33-1-4217-7491; E-mail: KORNER{at}ccr.jussieu.fr.
1
The abbreviations used are: NF-B, nuclear
factor
B; IL, interleukin; TNF, tumor necrosis factor; ICAM-1,
intercellular adhesion molecule-1; HIV, human immunodeficiency virus;
CMV, cytomegalovirus; EMSA, electrophoretic shift assay; LTR, long
terminal repeat; CAT, chloramphenicol acetyltransferase; Luc,
luciferase; PMA, phorbol 12-myristate 13-acetate; ELISA, enzyme-linked
immunosorbent assay; PHA, phytohemagglutinin.
2 J. Feuillard and M. Körner, unpublished observation.
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REFERENCES |
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