(Received for publication, May 22, 1995; and in revised form, September 1, 1995)
From the
We investigated the molecular basis of the synergistic induction
by interferon- (IFN-
)/tumor necrosis factor-
(TNF-
)
of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and
compared it with the basis of this induction by lipopolysaccharide
(LPS). Functional studies with IL-6 promoter demonstrated that three
regions are the targets of the IFN-
and/or TNF-
action,
whereas only one of these regions seemed to be implicated in LPS
activation. The three regions concerned are: 1) a region between
-73 and -36, which is the minimal element inducible by LPS
or TNF-
; 2) an element located between -181 and -73,
which appeared to regulate the response to IFN-
and TNF-
negatively; and 3) a distal element upstream of -224, which was
inducible by IFN-
alone. LPS signaling was found to involve
NF
B activation by the p50/p65 heterodimers. Synergistic induction
of the IL-6 gene by IFN-
and TNF-
, in monocytic cells,
involved cooperation between the IRF-1 and NF
B p65 homodimers with
concomitant removal of the negative effect of the retinoblastoma
control element present in the IL-6 promoter. This removal occurred by
activation of the constitutive Sp1 factor, whose increased binding
activity and phosphorylation were mediated by IFN-
.
IL-6 is a multifunctional cytokine involved in
controlling many cell functions, including antibody synthesis by B
cells, T cell cytotoxicity, stem cell differentiation, and induction of
acute phase proteins. IL-6 is produced in response to a variety of
noxious stimuli, including viral and bacterial
infections(1, 2, 3, 4) . Deficient
regulation of the IL-6 gene is involved in the pathogenesis of
autoimmune diseases and affects normal and leukemic
hematopoiesis(4, 5, 6, 7, 8, 9) .
Recent studies demonstrated the presence of a defect in IL-6 production
in Fanconi's anemia(10, 11) , suggesting that is
partly responsible for the altered hemopoiesis in Fanconi's
anemia patients.
The transcriptional regulatory elements present in
the 5`-flanking region of the human IL-6 gene have been studied by
several laboratories. Various elements responsible for IL-6 gene
induction have been identified, including phorbol 12-myristate
13-acetate, cAMP, NFB, NF-IL-6, and multiple cytokine-responsive
elements(3, 4, 12, 13, 14, 15, 16) .
Tumor suppressor gene products p53 and pRB have been reported to
suppress IL-6 promoter activity(17) .
Different
laboratories, including ours, showed that there are cell type-
dependent differences in the mechanism and transcription factors
involved in IL-6 gene induction. Thus, TNF- induced IL-6 in a
variety of cell
types(18, 19, 20, 21) , but it
failed to do so in monocytes(2, 3, 20) . We
have shown that IFN-
is an essential co-signal for TNF-
in
the induction of IL-6 in monocytic THP-1 cells(22) .
The role of IL-6 in the immune response, acute phase reaction, and hematopoiesis, and the fact that monocytic cells appeared to be one of the major physiological sources of this cytokine, prompted us to analyze the molecular mechanism responsible for its induction in this cell type.
Although the mechanism of transcriptional activation
mediated by different inducers in several cell types has been
extensively studied, the mechanism responsible for synergistic IL-6
gene induction by IFN- and TNF-
in monocytic cells has still
not been identified.
We postulated that both IFN- and TNF-
activate transcription factors, which should act simultaneously to
induce IL-6 gene expression. This hypothesis was supported by our
previous finding that sequential stimulation with IFN-
, followed
by TNF-
stimulation, did not lead to IL-6 mRNA
induction(22) . This implied that IFN-
and TNF-
induced or activated different transiently expressed components, which
must act together in cooperation to trigger IL-6 gene expression.
It
has been suggested that the induction of NFB binding activity by
TNF-
contributes to the activation of the IL-6 promoter in some
cell lines, including monocytic
cells(3, 18, 19, 20) . The
NF
B/Rel family of transcription factors consists of at least five
proteins, including p65 (Rel A), p50, c-Rel, and p52, which are related
to each other through an N-terminal stretch of 300 amino acids called
the Rel homology domain. DNA binding occurs through dimerization of the
family members, resulting in numerous homo- and heterodimeric
combinations of NF
B/Rel proteins. The C-terminal region of p65,
c-Rel, and Rel-B harbor a transcriptional activation domain, and the in vivo transcriptional activity is attributed to the p65-,
c-Rel-, and Rel-B-containing dimers(23) .
The elements
involved in the IFN--mediated induction of IL-6 expression are not
known. The binding of IFN
/
to their specific receptors
rapidly activates a latent cytoplasmic transcription factor, ISGF3
(interferon-stimulated gene factor 3). ISGF3 is transiently activated
and has been shown to stimulate ISRE-dependent
transcription(24, 25, 26, 27) .
Other regulatory factors, including interferon-regulatory factor 1
(IRF-1) and IRF-2, have also been shown to be involved in the
regulation of the IFN system. IRF-1 and IRF-2 bind to similar cis
elements within type I IFN and IFN-inducible genes. IRF-1 functions as
a transcriptional activator, while IRF-2 represses IRF-1
function(28, 29, 30, 31, 32, 33) .
Both factors appear also to bind specifically to ISRE
sequences(34, 35) .
We performed a functional
analysis of the 5`-flanking region of IL-6 gene using transient
transfection of CAT reporter gene linked to IL-6 promoter, in order to
clarify the molecular mechanism involved in the synergistic induction
by IFN- and TNF-
of the IL-6 gene in human monocytes. The
monocytic THP-1 cell model appears to be particularly suitable for
molecular dissection of IFN-
/TNF-
-mediated synergistic
induction of IL-6 gene expression, because in these cells, the IL-6
gene is not constitutively expressed(22) .
Analysis of IL-6
promoter constructs, electrophoresis mobility shift assay (EMSA), and
immunoprecipitation analysis have shown that the synergistic induction
of IL-6 gene by IFN- and TNF-
in human monocytic cells
involved cooperation between IRF-1 and NF
B binding elements, as
well as the removal of the negative effect of the retinoblastoma
control element (RCE) present in the IL-6 promoter (IL-6-RCE). This
IL-6-RCE contained, among other components, a core 5`-CCGCC-3` sequence
homologous with the consensus binding motif, which is the target of the
Sp1 factor. Sp1 is a constitutively expressed transcription factor
present in a wide range of cell types and binds a GC-rich consensus
sequence present in many cellular and viral promoters. Sp1 contains
three zinc fingers that mediate DNA binding and four domains that
mediate transcriptional activation(36) . Upon binding to the
DNA containing the GC box in the cell nucleus, Sp1 becomes
phosphorylated on multiple sites by double-stranded DNA-dependent
kinase(37) . It has been shown that specific interaction
between DNA-binding domains of the p65 subunit-NF
B and Sp1 bound
the DNA modulates transcription of human immunodeficiency virus type 1
in response to cellular activation(38) .
Our results show
that the contribution of IFN- to the triggering of IL-6 gene
expression in human monocytes involves a change in the amount and
phosphorylated state of Sp1, together with the induction and activation
of IRF-1. These factors cooperate synergistically with homodimer
p65-NF
B, which is activated by TNF-
.
For EMSA, nuclear protein (10 µg) was
incubated with radiolabeled probe (20,000 cpm) in buffer containing 50
mM NaCl, 10 mM HEPES (pH 7.9), 5 mM Tris-HCl
(pH 7.9), 1 mM dithiothreitol, 15 mM EDTA, 10%
glycerol, 500 µg/ml BSA-FV, and 800 µg/ml denaturated salmon
sperm DNA (in a final volume of 12.5 µl) (without EDTA in the case
of SP1 oligonucleotide probe). After a 30-min incubation on ice, the
nucleoprotein complexes were resolved by a nondenaturing
electrophoresis in a 5% polyacrylamide gel for 3 h at 20 mA in 1
TGE buffer (50 mM Tris-HCl (pH 8.8), 180 mM glycine, and 2.5 mM EDTA), in refrigerated condition. The
gel was dried and exposed overnight to a PhosphorImager screen
(Molecular Dynamics, Sunnyvale, CA). For competition experiments, a
100-fold molar excess of the unlabeled oligonucleotides was added 15
min before incubation of nuclear extracts with the end-labeled
oligonucleotides, while antisera were mixed directly with nuclear
extracts and binding buffer (without salmon sperm DNA) 1 h before
adding salmon sperm DNA and radiolabeled probe.
Figure 1:
Functional analysis of the
5`-cis-regulatory elements of the IL-6 gene: relative CAT activity. A, schematic extended map of the IL-6 promoter, with the
locations of DNA motifs known to be implicated in IL-6 gene induction
by various inducers, and deletion mutants of the human IL-6 promoter
fused to the promoterless CAT gene. B, to assess basal
promoter activity and its responsiveness to IFN- and/or TNF-
,
and to LPS, THP-1 cells were tranfected as described under
``Materials and Methods.'' CAT activity was determined after
36 h. The IL-6 promoter response (-fold induction) is the ratio of CAT
activity in stimulated cells to that in unstimulated cells, which is
defined as 1.0. Values are means of eight independent experiments with
standard deviation less than 10%.
Although
successive deletions at the 5` end of the IL-6 promoter fragment
resulted in a progressive reduction of the inducibility of CAT activity
by LPS, the construct containing the region from -73 to +14
retained its sensitivity to LPS and continued to display a 3-fold
increase in CAT activity. It was interesting to observe that this
fragment became inducible by TNF- and exhibited a 2-fold increase
in CAT activity. However, no synergistic effect was induced by
IFN-
+ TNF-
. Removal of the region from -1200 to
-36 resulted in the complete loss of inducibility by IFN-
and/or TNF-
, and by LPS (Fig. 1B).
The results
of the deletion analysis described above suggested the presence of at
least three regions differentially involved in IL-6 gene regulation by
IFN- and/or TNF-
, and by LPS. The first region is a distal
fragment between -1200 and -224 that positively regulates
the response to IFN-
alone. Although deletion of this fragment
does not eliminate the synergistic IFN-
+ TNF-
response,
a slide reduction in the magnitude of the induction of CAT activity is
observed. Computer-based observation showed that this fragment contains
an AP-1 site between -283 and -277, and a copy of the IFN
enhancer core sequence 5`-AAAGGA-3`
(-253/-248)(13) . The second region is a repressor
element, i.e. the sequence between -181 and -73,
which would negatively affect the synergistic response to IFN-
and
TNF-
without affecting sensitivity to LPS. This IL-6 DNA sequence
contains, from -126 to -101, a direct repeat which is
strikingly similar to the c-fos basal transcription element,
with 21/26 nucleotides matching the RB-repressible RCE (the RB control
element) identified in the c-fos gene(17, 46) . In this connection, Santhanam et al.(17) showed in functional assays that the
overexpression of wild type RB in Hela cells strongly repressed the
activity of IL-6 promoter constructs. In addition to these two
elements, a third region located between -73 and -36,
probably corresponding to the minimal element necessary for the LPS
response, also allowed TNF-
sensitivity. Within this fragment were
a putative AP-1 motif (-61 to -55) and the sequence
5`-GGGATTTTCC-3` (-72 to -63), which is highly homologous
to the immunoglobulin
light-chain enhancer sequence
5`-GGGGACTTTCC-3`(42) .
Nuclear protein extracts, prepare
d from both untreated cells and
cells stimulated for 1 h with either IFN- and/or TNF-
or with
LPS, were submitted to EMSA with several radiolabeled double-stranded
synthetic oligonucleotides. This experiment was an initial experiment,
which was designed to screen the early induction of specific
protein-DNA complexes.
Fig. 2shows an EMSA using five
different oligonucleotides corresponding to (i) NFB-like and AP-1
binding motifs (A/-73), (ii) IL-6-RCE (RB control element) target
motif (B/-126), (iii) the typical cAMP/phorbol ester-responsive
motif, and the IL-1/TNF-
responsive elements (C/-173), (iv)
the negative regulatory domain-like sequence (and two putative copies
of GGAAA motifs considered to be responsible for IFN inducibility
(D/-207), and (v) the consensus AP-1 motif and a copy of the IFN
enhancer core sequence 5`-AAAGGA-3` (E/-283)(13) .
Figure 2: IFN- and/or TNF-
, and LPS
induce binding of specific nuclear proteins to the 5`-flanking region of
the human IL-6 gene. A representative EMSA analysis showed the specific
binding to oligonucleotide probes corresponding to IL-6 promoter
fragments: A, -73 to -54 (lanes 1-6);
B, -126 to -101 (lanes 7-13); C,
-173 to -145 (lanes 14-19); D, -207 to
-184 (lanes 20-25); E, -283 to -242
(lanes 26-32). To determine the binding specificity, nuclear
extracts from untreated THP-1 cells, or cells treated for 1 h with
IFN-
and/or
TNF-
or with LPS, were compared by EMSA: lanes 1, 7,
14, 20, and 26, untreated nuclear extracts; lanes
2, 8, 15, 21, and 27, IFN-
-treated nuclear extracts;
lanes 3, 9, 16, 22, and 28, TNF-
-treated nuclear extracts; lanes 4, 10,
17, 23, and 29, IFN-
+ TNF-
-treated nuclear extracts; lanes 5, 11, 18, 24, and 30, LPS-treated
nuclear extracts. a* indicates competition with specific
unlabeled double-stranded oligonucleotides (lanes 7, 12, 18, 25, and 31), and b* indicates competition with unlabeled unrelated
double-stranded oligonucleotide (lanes 13 and 32).
Formation of protein-DNA complexes was apparent with the three regions A, B, and E described above, which were involved in the functional response of the 5`-flanking regions of the IL-6 gene (Fig. 2). Surprisingly, no specific complexes were observed with regions C and D, previously reported to be involved in IL-6 gene regulation by various inducers in other cells systems(3, 4, 12, 13, 14, 15, 16) .
Figure 3:
Kinetics of induction of NFB-binding
protein in THP-1 cells. A, THP-1 cells were stimulated with
IFN-
and/or TNF-
, with IFN-
, or wi
th LPS. Nuclear
extracts were prepared after cell stimulation for 0.5, 1, or 2 h. EMSA
was performed using radiolabeled oligonucleotide A/-73. B, specificity of protein-DNA complexes. EMSA was performed
with nuclear extracts from untreated cells (Cont) or cells
stimulated for 2 h with TNF-
or LPS. Competition studies were
carried out with specific oligonucleotides A/-73 (-73) and consensus NF
B, or with unrelated
oligonucleotides AP-1, B/-126 (-126), or Sp1. In
addition, binding studies including antibodies were carried out with
two purified rabbit antisera, specifically reactive with p50 or p65
NF
B subunits (Ab p50, p65). NI,
nonimmune antiserum added for nonspecific reactions. C,
nuclear extracts from THP-1 cells, either untreated (Cont) or
treated with various inducers for 2 h, were submitted to
immunoprecipitation using either specific antibodies against p50 or p65
NF
B subunits, or nonimmune serum. After Western blot analysis,
specific proteins were revealed with the corresponding antibodies, as
described under ``Materials and Methods.'' Arrows point to specific p65 (left) or p50 (right)
NF
B subunits. The relative mass of protein molecular markers is
shown in the middle (Rainbow(TM),
Amersham).
Polyclonal
rabbit antibodies against p50 and p65 subunits of NFB were used to
probe the nuclear IL-6-NF
B binding complexes for the presence of
corresponding proteins (Fig. 3B). Addition of antisera
against the p65-NF
B subunit supershifted the protein-DNA complexes
induced by TNF-
but impaired the formation of the complexes
induced by LPS. Addition of antisera against the NF
B subunit p50
did not modify the TNF-
-induced complexes, but those induced by
LPS were partially supershifted. Antibodies against the p52 and c-Rel
NF
B subunits did not interact with any of these protein-DNA
complexes. Nevertheless, combined treatment with antibodies against p65
+ p52 + c-Rel NF
B subunits appeared to result in the
disappearance of the minor residual complex left in TNF-
-treated
cell extracts (data not shown). Results similar to those shown for the
TNF-
-induced complexes were obtained using nuclear extracts of
cells stimulated by IFN-
+ TNF-
. These results suggest
that p65 is contained in the complexes, whatever the inducers used,
whereas p50 only seems to be a constituent of the protein-DNA complexes
induced by LPS.
To further investigate the subunit composition of
the nuclear IL-6-NFB binding complexes, nuclear extracts were
immunoprecipitated with specific antibodies, and analyzed by Western
blot (Fig. 3C). In agreement with the results shown in Fig. 3B, p65 subunit-NF
B was found afte
r
stimulation with TNF-
alone, with IFN-
+ TNF-
and
LPS, whereas antibody against p50 only revealed a major protein of 50
kDa after LPS stimulation (Fig. 3C).
We previously
showed that in THP-1 cells, stimulation by combined treatment with
IFN- and TNF-
was required for endogenous IL-6 gene
expression and protein secretion, and that TNF-
treatment alone
was ineffective(22) . Here, however, TNF-
was effective in
activating nuclear protein binding to the NF
B-like element and in
driving CAT expression after transfection with the del(-73)
construct containing the NF
B-like sequence. These observations
suggested the presence of a repressive element(s) that might negatively
affect TNF-
-mediated transcription of the endogenous IL-6 gene.
The results shown in Fig. 1suggest that a cis-DNA element, which is located between -181 and -73 and contains, among others, the RCE motifs, might be involved in repression of IL-6 gene expression in THP-1 cells. These observations prompted us to investigate the interactions between proteins and the IL-6-RCE sequence in nuclear extracts of THP-1 cells stimulated with various inducers. For this investigation, we used an oligonucleotide spanning both sites (B/-126). EMSA revealed the presence of a major constitutive complex in untreated cells (Fig. 4A, left section). No major modification was observed after this stimulation. Complex formation was specific, since it competed with the non-radiolabeled specific oligonucleotide but not with the unrelated oligonucleotide AP-1 (Fig. 4B). Note that protein-DNA interactions appeared to involve the Sp1 elements, since binding displayed complete competition with a Sp1 consensus motif (Fig. 4B).
Figure 4:
Specificity of protein binding to RB
control elements. A, EMSA was performed with nuclear extracts
of THP-1 cells, either untreated or stimulated for 0.5 h with IFN-
and/or TNF-
, LPS, or IFN-
, using radiolabeled double-stranded
oligonucleotide B/-126 (left section), or radiolabeled
consensus Sp1 oligonucleotide (right section). B,
nuclear extracts of cells left untreated for 0.5 h (Cont) were
submitted to EMSA using the B/-126 radiolabeled probe to reveal
specific competition between RCE-binding proteins and either unlabeled
double-stranded oligonucleotide B/-126 or Sp1 consensus
oligonucleotide. AP-1 unlabeled oligonucleotide was used as unrelated
competitor. Nuclear extracts of untreated cells (Cont) or
IFN-
-treated cells were preincubated with antibodies (Ab)
against Rb protein (Rb) or against Sp1 protein (Sp1),
and radiolabeled probe B/-126 was then added. Recombinant Sp1
protein (1.0 footprinting units) was mixed with nuclear extract of
untreated cells (Cont) or cells treated with IFN-
, and
radiolabeled B/-126 probe was then added (right
section). C, specifity of the EMSA complexes revealed
with radiolabeled probe Sp1. Before the addition of radiolabeled Sp1
oligonucleotide, nuclear extracts of untreated cells or cells treated
with IFN-
, TNF-
, or IFN-
+ TNF-
were mixed
either with unlabeled oligonucleotide (-126, Sp1), or with
antibodies (Ab) against Sp1 protein or Rb protein; NI corresponded to nonimmune rabbit antiserum. Sp1 protein (1.0
footprinting unit) was added to the same nuclear extracts. D,
immunoprecipitation of nuclear extracts of untreated THP-1 cells (Cont) or cells stimulated with IFN-
alone or IFN-
+ TNF-
, using antibodies against Sp1 protein, followed by
Western blot analysis. Upper part shows blot hybridization
with Sp1 antibodies. Lower part, the same blot was
dehybridized and reprobed with antibodies against phosphoserine
protein. Left arrows point to the specific Sp1 protein. Right arrows (mass in kDa) point to the relative mass of
protein molecular markers.
Antibodies against purified Sp1 protein partially supershifted the B/-126 complex, whereas antibodies against Rb protein had no effect on complex formation (Fig. 4B). Furthermore, addition of purified Sp1 protein to the nuclear extracts led to a higher site occupancy without changing mobility (Fig. 4B, right section).
Taken together,
these results suggest that Sp1 is part of the protein-DNA complex
formed with B/-126 oligonucleotide. This oligonucleotide, which
encompasses the RCE-IL-6 promoter element, includes the core sequence
5`-CCGCC-3` (-119 to -115), which covers part of the RCE
motif (-123 to -118); this motif appears to be homologous
with the 5`-CCGCCC-3` core sequence, reported to be the consensus
binding motif involved in the interaction with the Sp1 factor.
Furthermore, when we used an Sp1 consensus oligonucleotide as the
radiolabeled DNA probe in EMSA experiments, a major constitutive
complex was revealed (Fig. 4A, right section)
with a mobility similar to that obtained with the B/-126 probe (Fig. 4A, left section). The amount of the
protein-Sp1 oligonucleotide complex rose slightly after stimulation by
IFN-, TNF-
, LPS, or IFN-
, and rose
more markedly after
combined stimulation with IFN-
+ TNF-
. Complex formation
was impaired by competition with the specific unlabeled Sp1
oligonucleotide, and partial competition was observed with the
B/-126 oligonucleotide (Fig. 4C). As expected,
antibodies against the Sp1 protein led to a supershift of the
protein-DNA complex, whereas no effect was observed with pRb
antibodies. In the same way, as for the B/-126 probe complex,
addition of Sp1 protein greatly increased the amount of protein-DNA
complex revealed with the Sp1 oligonucleotide probe.
To demonstrate
the presence of Sp1 protein in the B/-126 DNA protein complex, we
used shift-Western blot analysis, with either the B/-126 probe or
the Sp1 consensus probe. When a complex was formed with the
B/-126 probe (Fig. 5, A1), we could not reveal
the presence of Rb protein (A2), whereas the antibodies
against Sp1 protein recognized a specific protein whose abundance
increased slightly after IFN- + TNF-
treatment (A3). The antibodies against Rb protein identified a fain
t
lower band, probably corresponding to free Rb protein (Fig. 5, A2).
Figure 5: Sp1 protein is part of IL-6-RCE-protein complex. EMSA was performed using either the radiolabeled B/-126 oligonucleotide (A) or the radiolabeled Sp1 consensus oligonucleotide (B). Shift-Western blot analysis was then performed as described under ``Materials and Methods.'' Radiolabeled DNA was visualized on DEAE membrane (A1 and B1) whereas either Rb protein (A2 and B2) or Sp1 protein (A3 and B3) was revealed on nitrocellulose by Western blot analysis using the respective specific antibodies against Rb protein and Sp1 protein.
As expected, in a similar experiment using a
radiolabeled Sp1 probe (Fig. 5B), antibodies against
Sp1 protein revealed a specific protein contained in the protein-DNA
complex (B3); the amount of Sp1 protein correlated with the
amount of DNA probe bound to this complex (B1), which
increased after IFN- + TNF-
treatment (B3). A
faint lower band was also seen, probably corresponding to free Sp1
protein. With pRb antibodies, no protein was revealed in the
protein-DNA complex, and as in the experiment using the B/-126
probe, a lower band was detected, corresponding to free Rb protein (Fig. 5, B2).
Immunoprecipitation of crude nuclear
extracts of THP-1 cells stimulated with IFN- alone or combined to
TNF-
, using antibodies against Sp1 protein, dramatically raised
the level of the 95-kDa native Sp1 protein as shown by Western blot
analysis (Fig. 4D, upper section). To
demonstrate that the effect of IFN-
or IFN-
+ TNF-
correlated with the activation of Sp1 protein, the same immuno-Western
blot was hybridized with a monoclonal anti-phosphoserine antibody. Fig. 4D (lower section) shows an increase in
phosphorylated Sp1 protein, previously reported to be the active form
of this nuclear factor(36) . These results correlate well with
the increase in binding activity shown by EMSA performed with the Sp1
probe after THP-1 cell treatment with IFN-
+ TNF-
(Fig. 4A, right s
ection).
Taken together, these results suggest the involvement of the Sp1 protein in the protein-DNA interaction of the RCE present in the IL-6 promoter. Although we cannot exclude the possibility that other factors interact with the RCE-IL-6 promoter, shift-Western blot and antibody-supershift experiments nevertheless suggest that Sp1 is part of the activity that interacts with this region.
Figure 6:
Induction and specificity of IFN-
regulated IRF-1/IRF-2 complexes. Radiolabeled double-stranded
oligonucleotide E/-283 was used for EMSA. A, binding
reactions were tested with nuclear extracts prepared from untreated
THP-1 cells or cells treated with various inducers for 0.5, 1, or 2 h. Arrows indicate C1- and C2-specific protein-DNA complexes. B, nuclear extracts from THP-1 cells treated for 2 h with
IFN-
+ TNF-
were used to determine the specificity of
the two complexes by means of the following unlabeled double-stranded
oligonucleotides: E/-283 without mutation (-283),
or with mutation (mt) in the AP-1 motif (mt 15) or in
the IFN enhancer core sequence (mt 31), ISRE, and C3.
Oligonucleotides AP-1, B/-126(-126) and Sp1 were used as
nonspecific competitors. C, before addition of radiolabeled
probe E/-283, nuclear extracts from THP-1 cells treated with
IFN-
+ TNF-
for 2 h were preincubated with antibodies
against IRF-2, IRF-1, or nonimmune antiserum (NI).
To define the sequence specificity of the C1 and C2 complexes, competition experiments were performed with synthetic oligonucleotides representing several mutations of the -283 to -242 region either in the putative interferon enhancer core sequence 5`-AAAGG-3` (-283/mt31) or in the AP1 motif (-283/mt15), and also with the ISRE of the 2`,5`-oligo(A) synthetase gene promoter, known to be implicated in IFN responses(43) , and with the C3 oligonucleotide defined by its IRF-1-binding activity(44) .
Oligonucleotide E/-283 and the mutant -283/mt15 competed for the formation of both complexes (Fig. 6B, mt 15), whereas the mutant -283/mt31 lost this capacity (Fig. 6B, mt 31), indicating that the IFN enhancer core sequence 5`-AAAGG-3` was involved in the DNA-protein interaction. Complete competition for the C1 and C2 complexes was observed with the ISRE and C3 oligonucleotides. A trimer AP-1 motif was not able to compete for either complex, in agreement with the behavior of mutant -283/mt15. No competition was observed neither with the Sp1 nor with B/-126 oligonucleotides (Fig. 6B).
To
investigate the possible relationship between the C1 and C2 complexes
and ISGF3(25, 26, 27, 47) , nuclear
extracts of THP-1 cells treated for 2 h with IFN- were tested for
their binding activity, using the E/-283, ISRE, or C3
oligonucleotides as DNA radiolabeled probes (Fig. 7). The
pattern of the protein-DNA complexes was very similar with the
E/-283 and C3 oligonucleotide probes, suggesting that they bind
the IRF-1-related factor. For ISRE-DNA-protein binding, only IFN-
treatment resulted in a double upper-induced binding activity (Fig. 7); several constitutive complexes showed no quantitative
or qualitative changes, whatever the inducers used. These results
suggest that C1 and C2 complexes did not involve ISGF3, but rather
IRF-1/IRF-2, unlike the IFN-
-induced complexes revealed with the
ISRE probe.
Figure 7:
E/-283, C3-radiolabeled
oligonucleotides, and ISRE-radiolabeled oligonucleotide gave different
patterns of the protein-DNA complexes. Nuclear extracts of THP-1 cells
treated for 2 h with IFN-, TNF-
, IFN-
+ TNF-
,
LPS, or IFN-
were tested for their binding activity, using
oligonucleotides radiolabeled with E/-283 (left
section), C3 (middle section), or ISRE (right
section). Double arrows indicate specific protein-DNA
complexes.
As shown in Fig. 6C, the amount of constitutive DNA-binding C1 complex was markedly reduced by the IRF-2 antibodies, while the inducible complex C2 was supershifted by the IRF-1 antibodies. The IRF-1 antibodies also abolished the time-dependent increase in the C1 complex, which remained at its constitutive level. These results demonstrate that IRF-2 is involved in the formation of the constitutive C1 complex, and that IRF-1 is involved both in the formation of the inducible C2 complex and in the increase in the amount of C1 complex.
To establish whether this
typical IRF recognition sequence is sufficient for IFN- activation
and for the synergistic action of IFN-
with TNF-
, we
constructed mutated IL-6 promoter CAT expression plasmids. For this
purpose, we linked oligonucleotide E/-283, or oligonucleotide
-283/mt31, which carried a mutation in the putative IFN enhancer
core sequence AAAGGA (-253 to -248), to del(-224)-CAT
and called the resulting constructs del(-224)/E and
del(-224)/Emt31, respectively (Fig. 1A). These
constructs were then transfected into THP-1 cells. As shown in Fig. 1B, the relative induction of CAT activity after
THP-1 transfection with construct del(-224)/E demonstrated that
the IFN enhancer core sequence is sufficient to confer responsiveness
to IFN-
, because the mutation of the AAAGGA motif (see
del(-224)/Emt31) abolished IFN-
sensitivity; the IFN-
+ TNF-
synergistic induction was preserved, although to a
much smaller extent (2-fold induction). These results suggest that in
addition to the IFN enhancer core sequence, surrounding sequences might
contribute to the synergistic effect of IFN-
with TNF-
.
Transfection of either of the constructs del( -224)/E or del(-224)/mt31 resulted in a similar LPS-induced increase in CAT activity (7- and 6-fold, respectively), showing that the IFN enhancer core sequence did not play an essential role in LPS induction (Fig. 1). However, LPS inducibility required the cooperation of other(s) factor(s), as demonstrated by the progressive reduction of CAT activity after the transfection of IL-6 promoter 5` end successive deletions.
These results showed that IFN- + TNF-
and
LPS induced the IL-6 gene by different pathways.
For this purpose immuno-Western
blot analysis of nuclear extracts of THP-1 cells after 2 h of
stimulation was performed using antibodies against IRF-1 or IRF-2. A
polypeptide of 42 kDa was revealed by the IRF-1 antibodies, but only
after stimulation by IFN- or IFN-
+ TNF-
(Fig. 8A, right section). The IRF-2 antibodies
detected a constitutive IRF-2 protein whose abundance was not modified
after cell treatment with IFN-
or TNF-
either alone or
combined for 2 h. In contrast, treatment with LPS or IFN-
reduced
the amount of IRF-2 protein (Fig. 8A, left
section). Since formation of the constitutive C1 complex involved
IRF-2 protein binding (see Fig. 6C), we attempted to
establish whether a transient modulation of this protein occurred soon
after stimulation. As shown in Fig. 8A (left
section), treatment with IFN-
+ TNF-
, LPS, or
IFN-
reduced the level of IRF-2 protein in nuclear extracts of
cells stimulated for 30 min. The effect of IFN-
+TNF-
was
transient, because the reduction in the amount of IRF-2 protein was not
seen in nuclear extracts of cells that had been stimulated for 2 h. In
contrast, the inhibition by IFN-
or LPS of the IRF-2 protein level
lasted throughout the 2 h of stimulation.
Figure 8:
Modulation of IRF-1 or IRF-2 proteins. A, nuclear extracts of untreated THP-1 cells (CONT)
or cells treated with various inducers for 30 min (upper
section) or for 2 h (lower section) were submitted to
immunoprecipitation using specific antibodies against IRF-1 or IRF-2,
followed by Western blot analysis. IRF-2 protein was analyzed after
treatment of THP-1 cells for 30 min or 2 h by various inducers, whereas
IRF-1 protein was analyzed after cell stimulation for 2 h. Middle
arrows indicate the relative mass of protein molecular weight
markers. Left or right external arrows indicate
specific IRF-2 or IRF-1 proteins. B, in vitro translation: total RNAs were extracted from cells left untreated
for 2 h (Cont) or stimulated for 2 h, and were translated in vitro at a concentration of 480 µg total RNA/ml, using
reticulocyte lysate and [S]methionine under
defined optimal conditions (97 mM KCl and 1.8 mM
Mg(CH
COO)
). After incubation of 1 h at 32
°C, volume aliquots corresponding to 5
10
cpm
of labeled polypeptides were submitted to immunoprecipitation followed
by Western blot analysis, as described under ``Materials and
Methods.''
Since the amount of IRF-2
protein diminished after cell stimulation by IFN- +
TNF-
, LPS, or IFN-
, whereas IRF-1 protein was only induced by
IFN-
, we explored this effect to see if it was related to an
increase in IRF-1 and (or) a decrease in IRF-2 gene expressions. For
this purpose, total RNAs were extracted after 0.5, 1, or 2 h from cells
stimulated with various inducers and then analyzed by Northern blot. As
shown in Fig. 9, IFN-
already induced IRF-1 mRNA after 30
min, in an amount which increased for up to 2 h of cell stimulation. No
synergistic effect was observed after combined treatment with IFN-
and TNF-
. Stimulation by IFN-
also triggered IRF-1 gene
expression, but with different kinetics of accumulation since IRF-1
mRNA was barely detected after 1 h of IFN-
stimulation but its
amount increased thereafter. Two IRF-1 mRNAs of 2.1 and 3 kilobases
were revealed, with a similar accumulation kinetic. Neither TNF-
nor LPS induced IRF-1 gene expression in THP-1 cells.
Figure 9: Kinetics of IRF-1 and IRF-2 gene expression in THP-1 cells. Total RNAs (15 µg) of THP-1 cells treated with the indicated inducers for 0.5, 1, or 2 h were analyzed by Northern blot hybridization with the IRF-1-radiolabeled probe. After dehybridization, the same membrane transfer was successively hybridized with IRF-2 and actin probes. Arrows indicate specific mRNA.
In contrast,
IRF-2 mRNA was constitutively expressed (Fig. 9). No significant
modification of the IRF-2 mRNA level was observed whatever the period
of incubation and the inducers used. The reduction in the level of this
protein after stimulations with various inducers, without concomitant
decrease in the IRF-2 mRNA level, prompted us to make sure that IRF-2
mRNA was functionally active. Accordingly, total RNAs were extracted
from cells left untreated from 2 h or stimulated for 2 h and translated in vitro, using reticulocyte lysate and
[S]methionine. Identical number of counts/min (5
10
cpm) were analyzed by immuno-Western blot, using
specific IRF-2 antibodies. Similar amounts of IRF-2 protein were
detected whatever the inducers used (Fig. 8B).
These
results indicate that the differential regulation of IRF-2 protein by
IFN-, LPS, and IFN-
takes place at post-translational level.
We investigated the molecular basis for the
IFN-/TNF-
-mediated synergistic induction of the human IL-6
gene in the THP-1 monocytic cell line by functional analysis of this
gene's promoter. The results provided evidence that three regions
inside this promoter are the targets of the IFN-
/TNF-
action:
(i) a region between -73 and -36, which constitutes the
minimal element inducible by LPS or TNF-
; (ii) an element located
between -181 and -73, which appears to regulate negatively
the response to IFN-
and TNF-
; and (iii) a distal element
upstream of -224, which is the only one inducible by IFN-
alone.
Each of the DNA elements corresponding to the three
functional regions inducible by IFN- and/or TNF-
and by LPS
led to the formation of specific DNA-protein complexes.
The IL-6
promoter DNA fragment between -73 and -36 was found to
contain an NFB-like element near a putative AP-1 binding motif. In
THP-1 cells, TNF-
or LPS induced binding to this DNA motif,
whereas IFN-
neither induced NF
B binding activity nor
amplified the effect of TNF-
when added together with it. The
EMSA-DNA competition experiments with consensus immunoglobulin-
B
oligonucleotide allowed us to conclude that the formation of the
complexes induced by TNF-
and LPS is due to NF
B binding
factors. EMSA including antibodies and immunoprecipitation with
anti-p50 and anti-p65 NF
B subunit antibodies revealed only the
nuclear accumulation of p50 and p65 after stimulation by LPS. The
predominance of the p65 subunit was shown to be the major factor
induced by TNF-
or IFN-
+ TNF-
.
Various dimer
combinations of induced factors have been reported to be
stimulus-dependent and to display distinct DNA binding specificities by
activating distinct sets ot target genes(23) . Thus, homodimer
p65 was previously shown to be selectively activated in other systems
by TNF-
(48, 49) .
In THP-1 cells, the
induction of NFB binding activity by TNF-
did not correlate
with concomitant IL-6 production(22) , but required the
addition of IFN-
, unlike activation by LPS. This indicated that
the mechanism involved in the induction of endogenous IL-6 gene in
THP-1 cells is inducer-specific.
Although NFB binding factors
are certainly involved in IL-6 gene induction, the functional analysis
of IL-6-CAT construct deletions reported here shows that significantly
greater stimulation of the CAT reporter gene is obtained for constructs
that contain the 5` distal sequence of IL-6 promoter (pr-1200).
Successive deletions of the 5` end of the IL-6 promoter fragment
(-1200 to -108) resulted in the gradual reduction of
synergistic CAT activity by IFN-
+ TNF-
.
EMSA using
the oligonucleotide E/-283, which contains a copy of
IFN-enhancer-core sequence, showed that IFN-, but not TNF-
or
LPS, induced specific binding activity. Similar results were obtained
with C3 oligonucleotide, suggesting that an IRF-1-related factor was a
component of the C1- and C2-specific protein-DNA complexes. EMSA
experiments using antibodies against IRF-1 or IRF-2 allowed us to
conclude that the formation of the constitutive C1 complex involved
IRF-2 binding and that IRF-1 was involved, both in the binding activity
of the inducible C2 complex and in the increased abundance of the C1
complex due to stimulation by IFN-
.
In agreement with these
findings, transfection of construct del(-224)/Emt31 into THP-1
cells confirmed that the IFN-enhancer-core sequence AAAGG, which is
located between -253 and -248(13) , is sufficient
to induce the response to IFN- alone.
The IRF-1 binding site
identified in the IL-6 promoter was shown to be the central motif of
the IFN-stimulated response element (ISRE) found in the promoter of
IFN--stimulated genes such as the 2`,5`-oligo(A)-synthetase
gene(43) . In EMSA using the E/-283 probe, the
IRF-related complexes C1 and C2 were impaired by the ISRE
oligonucleotide. However, after IFN-
treatment, no C1 or C2
binding activity was observed. When EMSA was performed with the ISRE
oligonucleotide for purposes of comparison, it only revealed
ISGF3-related complexes after IFN-
treatment, as expected (24, 25, 26, 27) .
Induction of
IRF-1 protein-DNA binding by IFN- correlated with the induction of
IRF-1 mRNA and with the concomitant IRF-1 protein synthesis. On the
other hand, IRF-2 mRNA constitutive expression was not modified
whatever the inducers, whereas the level of constitutive IRF-2 protein
decreased transiently after IFN-
+ TNF-
treatment. LPS
or IFN-
treatment led to the disappearance of the constitutive
IRF-2 protein.
The different effects exerted on IRF-2 protein
down-regulation by IFN- + TNF-
on the one hand and by
LPS on the other might partly account for the difference between the
magnitude of IL-6 induction by each of these inducers in monocytic
THP-1 cells.
LPS was recently shown to induce or activate proteins
that recognize the 3`-end instability sequence of an mRNA that prevents
the latter's translation(50, 51, 52) .
Such a mechanism might account for the down-regulation of IRF-2 protein in vivo, since here we were unable to show by Northern blot
analysis that this inducer modulates either the expression of mRNA
IRF-2 or its translation in vitro. Alternatively, the
constitutive IRF-2 protein might be rapidly degraded after its
activation by IFN- or LPS, as shown in other systems(53) ,
thus allowing the binding of another activator which in the case of
IFN-
would be IRF-1.
As LPS did not, under our experimental conditions, induce IRF-1, we might be justified in assuming that one or several other factors activated by LPS could act as a transcriptional factor, as suggested by our functional CAT analysis (Fig. 1B).
Our results suggest that IRF-1 may be a
critical downstream signaling factor involved in IFN- signal
transduction in monocytes/macrophages, particularly for genes whose
maximal expression is triggered by combined treatment with IFN-
+ TNF-
(22, 54, 55, 56) .
The fact that TNF-
induced NF
B binding activity, which
correlated with the inducibility of the del(-73)-CAT construct
but failed to induce reporter constructs containing additional upstream
sequences, suggests that a silencer of NF
B activity must be
present in the IL-6 promoter. Functional analysis of deletion
constructs indicated that this negative element might be located
between -181 and -73. This region contains, among others, a
retinoblastoma control element known to be involved in pRb-mediated
repression of the c-fos promoter(46) . The presence,
as reported by Santhanam et al., of a region analogous to the
RCE in the IL-6 promoter between positions -126/-101,
exhibiting similar patterns of c-fos and IL-6 promoters
regulation, suggests that RCE may be involved in IL-6 gene repression
in monocytic cells. Evidence that RCE has a repressor role was
obtained, in NIH 3T3 cells, by deletion experiments and cotransfections
of IL-6 promoter with pRB expressing vector (17) .
It is
tempting to suggest that alteration(s) in the interaction between the
putative suppressor factor(s) and RCE allowed us to obtain a
synergistic effect of IFN- with TNF-
in THP-1 cells.
Synergistic CAT induction was observed for del(-181) but not for
del(-108). The del(-181)-CAT construct contains the intact
IL-6-RCE motif, whereas a truncated RCE motif is present in the
del(-108)-CAT construct.
EMSA experiments performed with oligonucleotide B/-126, which contains the IL-6-RCE motif, revealed one major constitutive specific RCE-protein complex in nuclear extracts of THP-1 cells. This complex was completely inhibited by consensus Sp1 oligonucleotide and was supershifted by Sp1 antibodies. In addition, recombinant Sp1 protein increased this site occupancy. Furthermore, we showed by shift-Western blot analysis that Sp1 protein is part of the IL-6-RCE-protein complex, whereas Rb protein, undetectable in this complex, was not.
Our observations are in agreement with the hypothesis proposed by Udvadia et al. that Rb regulates transcription partly by virtue of its ability to interact functionally with RB control proteins, including Sp1(45, 57, 58, 59, 60) .
EMSA performed with the Sp1 consensus probe showed that stimulation
of THP-1 cells by IFN- + TNF-
enhanced the amount of the
protein-DNA complex. This result correlates with a marked increase by
IFN-
in the amount of serine-phosphorylated Sp1 protein.
It has been shown that Sp1 is selectively phosphorylated upon binding to its cognate recognition elements on DNA, suggesting that phosphorylation represents an early event in the processes leading to transcriptional activation by Sp1. Phosphorylation of Sp1 is catalyzed by a double-stranded DNA-dependent kinase and requires binding to DNA containing the GC box(34, 61) .
It is tempting to
postulate that the effect of IFN- was partly mediated by increased
binding of Sp1 to its target sequence in the IL-6 promoter, with
concomitant Sp1 factor activation by specific phosphorylation. This
alteration might impair the negative effect mediated by the IL-6-RCE.
On the other hand, NFB has been shown to synergize with a
number of transcription factors, including
Sp1(62, 63) . It has been reported that Sp1
specifically interacts with the N-terminal region of Rel A (p65);
similarly, Rel A (p65) bound directly to the zinc finger region of Sp1
factor. This interaction is specific, because Rel A did not associate
with several other transcription factors known to be zinc finger
proteins(36, 37, 38) . Since the p65
homodimer induced by TNF-
in THP-1 cells is not, by itself,
sufficient to induce IL-6 gene expression, we can postulate that
functional interaction between p65 NF
B and the activated-Sp1 bound
to the adjacent target Sp1 site contributed to the induction of the
IL-6 gene.
Our observations allowed us to conclude that IL-6 gene
expression in THP-1 cells by IFN- + TNF-
, or by LPS
treatment is mediated through different signaling pathways.
LPS
signaling was shown to involve interaction with
CD14(64, 65) , which triggered the mitogen-activated
protein kinase Ras-Raf1-dependent cascade, leading to IB
phosphorylation and subsequent translocation allowing accumulation of
the p50/p65 NF
B heterodimer in the
nucleus(23, 66, 67, 68) . Since this
p50/p65 complex was also identified in our cell system, a similar
pathway might be responsible for the IL-6 gene expression induced by
LPS treatment in THP-1 cells.
In contrast, the synergistic effect of
IFN- + TNF-
on IL-6 gene induction in THP-1 cells might
involve three simultaneous processes. The first process is the
activation of NF
B by TNF-
by a different pathway from that of
activation by LPS, leading to the activation of the p65 homodimer. This
possibility is supported by a recent report showing that, in murine
macrophages, TNF-
activates the mitogen-activated protein kinase
cascade in a mitogen-activated protein kinase kinase kinase-dependent
but c-Raf-1-independent fashion(69) . The second process is the
induction of IRF-1 by IFN-
through a signaling pathway involving
the activation of Jak1/Jak2 and Stat
1(70, 71, 72, 73, 74, 75) .
The third process is a concomitant change by IFN-
, in the state of
phosphorylation and abundance of the constitutive Sp1 nuclear factor
interacting with IL-6-RCE. The phosphoserine kinase implicated in the
signaling pathway leading to phosphorylation of Sp1, presumably
activated by IFN-
, remains to be investigated. Transcriptional
induction of the IL-6 gene might result from a coordinated effect
exerted by factor Sp1 together with IRF-1 and p65 homodimer-NF
B.
The fact that regulation by IFN- of
the IL-6 gene in human
monocytes involved IRF-1 may be more generally related to the tumor
suppressor/differentiation properties of
IFN-
(32, 76) . Deletion of a chromosomal segment
that contains the IRF-1 gene, mapped in chromosome 5q31.1, is very
often observed in human leukemia(77) . It is therefore possible
that an alteration in the balance of IRF-1/IRF-2 may impair optimal
physiological induction of IL-6 by IFN-
/TNF-
in the monocytic
cell compartment, leading to abnormal cell maturation and proliferation
in certain hematological disorders and to neoplasia.
Alterations in
IL-6 production such as overexpression in multiple myeloma and other
type of
cancers(2, 3, 4, 5, 6, 7, 8, 9 in monocytic cells
required the induction of IRF-1 and involved phosphorylated Sp1 protein
may be of physiological relevance, and one of the important homeostatic
properties of IFN-
within the cytokine network.