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on IL-6-Induced 
2-Macroglobulin Expression Is Due to Activation of NF-
B1
*
Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, Aachen, Germany; and
Klinik für Gastroenterologie, Hepatologie und Infektiologie, Medizinische Klinik der Heinrich-Heine Universität, Düsseldorf, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and STAT3 are transcription factors
well known to be activated by many stimuli and to mediate
transcriptional activation by binding to specific enhancer sequences.
In this study, it is analyzed how IL-1
inhibits IL-6-induced
transcriptional activation of the 
2-macroglobulin
promoter. It is shown that IL-1 prevents STAT3 binding to the two
STAT3-responsive sites within the 2-macroglobulin
promoter by association of IL-1-activated NF-B to this region.
The observation that inhibition of IL-6-induced transcriptional
activation of this promoter by IL-1 is reversed by cotransfection
with I-B provides evidence that NF-B activation by IL-1 is
responsible for inhibition of IL-6-mediated trans
activation of the 2-macroglobulin gene. Accordingly,
cotransfection of the NF-B subunits p50 or p65 themselves inhibited
activation of the 2-macroglobulin promoter by IL-6.
Introduction of point mutations in each of the two NF-B sites
overlapping the two STAT3 binding sites within the
2-macroglobulin promoter provides evidence that each of
these two sites counteracts transcriptional activation via STAT3. Most
interestingly, at least one functional NF-B consensus site is
essential for the IL-6-induced transcriptional activation of the
2-macroglobulin promoter. Additional data are provided
indicating that the activation of NF-B by IL-1 is also
responsible for the inhibition of other IL-6-inducible genes, such as
the 1-antichymotrypsin gene as well as the suppressor of
cytokine signaling 3 gene, suggesting a more general relevance of this
mechanism for transcriptional regulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inflammatory cascade underlying the acute-phase response is largely
controlled by the action of different mediators released under
inflammatory conditions. Upon activation, blood monocytes and tissue
macrophages release a set of primary inflammatory mediators such as
IL-1 and TNF-, thereby inducing the synthesis and secretion of
several secondary cytokines and chemokines such as IL-6 and IL-8 from
macrophages, monocytes, and local stromal cells. Recruitment of other
immune effector cells by chemotaxis then rapidly augments the local
inflammatory response to counteract the inflammatory stimulus and to
remove the cellular debris generated by any associated tissue damage
(1).
One of the most extensively studied responses to an acute inflammatory stimulus is the change in the hepatic synthetic profile of acute-phase proteins (APP).4 IL-6 has been recognized to be the major mediator involved in the regulation and maintenance of the synthesis of most of these acute-phase proteins in the liver (2, 3).
The action of IL-6 on hepatocytes is mainly mediated via the Janus
kinase (Jak)/STAT signal transduction cascade (4, 5), a
key signaling system, involved in the signal transduction of numerous
ILs, the IFNs, as well as a number of growth and differentiation
factors (6, 7). Binding of these ligands to their
appropriate receptors activates tyrosine kinases of the Jak family,
followed by tyrosine phosphorylation, dimerization, and nuclear
translocation of so-called STATs. In the nucleus, activated STAT dimers
bind to specific enhancer sequences and modulate transcription of
target genes. Many APP genes such as those coding for C-reactive
protein (human), 1-antichymotrypsin
(1ACT) (human),
2-macroglobulin (2M)
(rat), and LPS-binding protein (human and rat) have been identified to
be induced through STAT factors (8, 9, 10). The activation of
Jak1 and STAT3 has been shown to be crucial for the transcriptional
activation of these genes by IL-6 (4, 5, 11).
Furthermore, IL-6-stimulated gene induction can be modulated via other
signal transduction pathways. Thus, STAT binding sites are often in
close proximity to binding sites for other transcription factors such
as NF-IL-6 (12), NF-B (13), AP-1
(14, 15), and GR (16), making a cooperative
action of these factors with STATs in gene regulation most likely.
Moreover, in the promoters of the rat 2M gene
and the human 1ACT gene, STAT3 binding sites
are arranged as a tandem (17, 18), suggesting that
formation of multimers on clustered binding sites also represents a
regulatory step in STAT-dependent gene activation. However, the exact
function of these tandem motifs has yet not been uncovered.
On the other hand, recent results from several laboratories strongly
indicate that IL-6-induced signaling and transcriptional activation are
often modulated at the level of signal transduction upstream from the
respective transcription factors. For example, LPS and the
proinflammatory cytokine TNF- have been shown to inhibit
IL-6-mediated STAT3 activation. This inhibition is most likely due to
the induction of the de novo synthesis of the Jak inhibitor suppressor
of cytokine signaling 3 (SOCS3) both by LPS and TNF-
(19). A similar mechanism has been discovered for IFN-
signaling, in which LPS inhibits IFN--dependent STAT1 activation
also via the induction of SOCS3 (20).
IL-1 dose dependently inhibits the IL-6-induced synthesis and
secretion of APP such as 2M and fibrinogen in
hepatocytes in primary culture (21). However, the
underlying mechanism remained unclear.
Analyzing the mechanism of IL-1-mediated attenuation of IL-6
signal transduction, we show that NF-B activated by IL-1
counteracts IL-6-induced transcriptional activation of the
2M promoter most likely by counteracting STAT3
DNA binding to its respective binding region within the
2M promoter. Interestingly, although NF-B
itself clearly inhibits STAT3 action, our data give further evidence
that at least one intact NF-B binding site seems to be essential for
IL-6-induced STAT3-dependent transcriptional activation of the
2M promoter. In this context, activation of
NF-B is assumed to be of more general relevance for the inhibitory
effects of IL-1 on IL-6-induced gene induction, since data are
provided, suggesting that the activation of NF-B by IL-1 also
inhibits the induction of an IL-6-inducible
1ACT reporter construct and the IL-6-mediated
increase of SOCS3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes were purchased from Roche Molecular
Biochemicals (Mannheim, Germany); Taq polymerase was from
Hybaid (Heidelberg, Germany); oligonucleotides were obtained from
MWG-Biotech (Ebersberg, Germany); SN50 and SN50 M (22)
were from Calbiochem (Bad Soden, Germany); DMEM and DMEM/nut.mix F12
were from Life Technologies (Eggstein, Germany); FCS was from Seromed
(Berlin, Germany); human rIL-6 and soluble IL-6R gp80 were prepared as
described (23); the internal control plasmid DNA pCH110
was from Amersham Pharmacia Biotech (Uppsala, Sweden). Purified p50 was
from Promega (Madison, WI); the specific Ab against p65 was from
Upstate Biotechnology (Lake Placid, NY); and the Ab against STAT3
was kindly provided by W. Müller-Ester (Mainz, Germany).
Preparation, cultivation, and stimulation of cells
Isolated parenchymal cells were prepared from livers of 5- to 8-wk-old male Wistar rats by a collagenase perfusion technique. Cells were plated on collagen-coated culture dishes and maintained in Krebs-Henseleit medium supplemented with 6 mmol/L glucose in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After 2 h, medium was removed and the culture was continued for 24 h in DMEM containing 5% FCS, 0.1 mg/ml penicillin/streptomycin, 100 nmol/L insulin, 100 nmol/L dexamethasone, 30 nmol/L Na-selenite, and 1 µg/ml aprotinin.
The human hepatoma cells HepG2 were grown in DMEM/nut.mix F12 supplemented with 10% FCS, streptomycin (100 mg/L), and penicillin (60 mg/L). Medium was changed and adjusted to 5 ml 24 h before experiments were conducted.
Cells grown in a 100-mm dish were stimulated with IL-1 or IL-6 at
the concentrations indicated. SN50 and SN50 M were dissolved in sterile
water. Cells were preincubated with SN50 and SN50 M for 15 min at the
concentrations indicated in the figure legends. Nuclear extracts were
prepared as described by Andrews and Faller (24). Protein
concentration was determined with a Bio-Rad (Munich, Germany) protein
assay.
Electrophoretic mobility shift assay
EMSAs were performed as described previously (18).
The protein/DNA complexes were separated on a 4.5% polyacrylamide gel
containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris base, 20 mM
boric acid, 0.5 mM EDTA, pH 8) at 20 V/cm for 4 h. Gels were fixed
in 10% methanol, 10% acetic acid, and 80% water for 1 h, dried,
and autoradiographed. The double-stranded
32P-labeled oligonucleotides used for EMSA are
listed in Fig. 1
. In addition to those depicted in Fig. 1, the
following oligonucleotides were used: mutated m67SIE oligonucleotide
from the c-fos promoter (m67SIE:
5'-GATCCGGGAGGGATTTACGGGAAATGCTG-3') (25) and an
oligonucleotide comprising the proximal (p) STAT3 binding site from the
2M promoter optimized for the binding of
NF-B (pNF-B: 5'-GATCCTTCTGGGAATTCCTA-3') (26). For
competition assays, unlabeled probes were used at 5, 10, 20, and 50
molar excess to the radioactive labeled probes. For supershift
analyses, the nuclear extracts were preincubated with the respective Ab
at 4°C for 20 min before EMSA procedures. For in vitro binding assay
using purified NF-B p50, p50 protein was added in increasing amounts
from 7, 12.5, 25, 50, to 100 ng to the nuclear extracts of
IL-6-stimulated cells.
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Standard cloning procedures were performed as outlined by
Sambrook et al. (27). pGL32 M-215Luc contains the
promoter region -209 to +8 of the 2M gene
fused to the luciferase-encoding sequence and was described previously
(28). pGL3-hACT-359Luc contains the promoter region -379
to +25 of the 1ACT gene fused to the
luciferase-encoding sequence and was kindly provided by F. Horn
(Leipzig, Germany) and described previously (29).
Mutations in the 2M promoter reporter
construct were generated by PCR technique using appropriate
oligonucleotides. The sequences of all constructs were controlled by
sequencing using an ABI Prism automated sequencer (PerkinElmer,
Norwalk, CT). The different point mutations introduced into the
2M gene promoter fragment are summarized in
Fig. 1
.
Transfection procedure and reporter gene assay
For transfection of HepG2 cells, cells were grown on 60-mm
dishes to 30% confluency and transfected in DMEM supplemented with
10% FCS. Calcium phosphate precipitation was performed with 3 µg
reporter construct, 2 µg -galactosidase expression vector
(pCR3lacZ; Amersham Pharmacia Biotech), and 4.5 µg I-B-encoding
expression, as indicated in the figure legends. Transfections were
adjusted with control vectors to equal amounts of DNA. Cells were
incubated with the precipitate for 16 h, washed twice with PBS,
and let for additional 10–24 h in fresh medium. For reporter gene
assays, cells were stimulated for 16 h. Cell lysis and luciferase
assays were conducted using the luciferase kit (Promega), as described
by the manufacturer’s instructions. All expression experiments were
done at least in triplicate. Luciferase activity values were normalized
to transfection efficiency monitored by the cotransfected
-galactosidase expression vector. Error bars are SD.
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed using the Chromatin
Immunoprecipitation Assay kit from Upstate Biotechnology. A total of
8 x 106 rat primary hepatocytes was seeded
on 10-cm dishes and analyzed for NF-B DNA binding, according to the
manufacturers’ instructions. NF-B/DNA complexes were precipitated
with an Ab specific for the p65 subunit of NF-B (Santa Cruz
Biotechnology, Santa Cruz, CA). Using PCR reaction, the purified
chromatin precipitates were analyzed for the existence of the following
rat 2M promoter fragments: -12 to -419 and
-103 to -283.
Total RNA isolation and Northern blot analysis
Total RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany), as described by the manufacturer. A total of 10 µg total RNA was separated on 1% denaturing agarose gels and transferred to a NitroPlus transfer membrane (Micron Separations, Westboro, MA). The membranes were prehybridized for 2 h at 68°C in 10% dextran sulfate, 1 M NaCl, and 1% SDS, and hybridized overnight in the same solution with cDNA fragments labeled with Random Primed DNA Labeling Kit (Roche Molecular Biochemicals). Blots were exposed to Kodak X-OMAT AR-5 film at -70°C with intensifying screens.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibits IL-6-induced DNA binding of activated STAT3 by
competition with NF-B at overlapping NF-B/STAT3 binding sites of
the 2M promoter
Whereas IL-1 is regarded as a proinflammatory cytokine, IL-6
exhibits many antiinflammatory activities. We have described previously
that IL-1 counteracts the IL-6-induced synthesis of APP, such as
2M and fibrinogen (21). However,
the underlying molecular mechanism for this inhibitory effect of
IL-1 is still unclear. Since STAT3 has been shown to be the major
transcription factor involved in IL-6-induced APP synthesis
(30), we analyzed whether the IL-6-induced STAT3
activation in human hepatoma (HepG2) cells is affected by a
preincubation of these cells with IL-1 (Fig. 1). As shown in the upper
panel of Fig. 1, preincubation with IL-1 for 10 min did hardly
counteract STAT3 DNA binding to a STAT3-specific DNA probe deduced from
the c-fos promoter (25). Thus, the
IL-6-mediated activation of STATs is essentially unaffected by IL-1.
However, IL-6-induced STAT3 DNA binding to a DNA fragment that
corresponds to the sequence -193 to -147 of the
2M promoter, containing a tandem STAT3-binding
motif (Fig. 2A), was almost
completely abolished by preincubation with IL-1 (Fig. 1, lower
panel). Moreover, in parallel to the IL-1-dependent
disappearance of the STAT3/DNA complex, a faster migrating complex
appeared (indicated as (II) in Fig. 1, lower panel). These
data strongly suggest that the inhibitory effect of IL-1 on
IL-6-induced APP synthesis is rather due to a disturbed DNA binding of
activated STAT3 to specific APP promoter elements than due to an
inhibition of STAT3 activation. The observation that the disappearance
of the STAT3/DNA complex is accompanied by the appearance of a faster
migrating complex suggests that inhibition of IL-6-induced APP
synthesis by IL-1 might be due to another protein competing STAT3
DNA binding. Presently, we have no explanation for the nature of the
gel-shift band with intermediate mobility (open arrowhead). It is
important to note that authentic oligonucleotides (Fig. 2) and not
probes designed for optimal transcription factor binding were used in
the present studies; these native oligonucleotides form protein/DNA
complexes of lower affinity reflected in the much lower intensities in
the EMSA.
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, supershift analyses with Abs raised against
the NF-B subunit p65 or STAT3 were performed (Fig. 3A). Incubation of nuclear
extracts from IL-6-stimulated cells with a specific antiserum to STAT3
led to the disappearance of the slower migrating complex, whereas a
faster migrating band turned up (compare lanes 2 and
3). On the other hand, the faster migrating complex
disappeared when nuclear extracts from cells costimulated with IL-1
and IL-6 were incubated with an Ab specific for p65 (compare
lanes 5 and 7). Incubation with a STAT3-specific
Ab strongly enhanced the amount of this protein/DNA complex
(lane 6). These data indicate that the slower
migrating complex contains STAT3, whereas the faster one contains
NF-B. Furthermore, these data give clear evidence that both STAT3
and NF-B are capable to bind and to compete for binding to the
2M promoter fragment (-193 to -147). In line
with this is the observation that the two STAT3 binding sites within
the 2M promoter fragment used (Fig. 2A; distal and proximal sites) overlap with the consensus
sequence GGGRNNYYCC, in which R is a purine and Y is a pyrimidine
(31), representing putative binding sites for NF-B
(Fig. 2A). Our data further suggest that a basal amount of
NF-B, competent to bind the 2M promoter, is
present in IL-6-stimulated nuclear extracts, and that removal of the
competing STAT3 from the protein/DNA complex by specific Abs enables or
enforces NF-B binding to the promoter element (compare lanes
2 and 3 in Fig. 3A).
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B and STAT3 for binding to
the 2M promoter element, we tested in gel
retardation assays whether increasing amounts of the commercially
available NF-B subunit p50 eliminate STAT3 binding to the DNA
element. As shown in Fig. 3B, the increasing amount of
NF-B bound to the promoter element paralleled the loss of STAT3/DNA
complexes. This observation further urges the assumption that IL-1
exerts its inhibitory function on the IL-6-induced
2M expression by activating NF-B, which
competes with STAT3 for binding to the 2M
promoter. This conclusion is also supported by our observation that
IL-1 does not affect STAT3 DNA binding to the SIE probe (Fig. 1, upper panel) due to the lack of an NF-B binding
site.
I-B impairs the inhibitory effect of IL-1 on IL-6-induced
promoter activation, whereas overexpression of the NF-B subunits p65
and p50 inhibits IL-6-induced promoter activation
To test whether NF-B is responsible for the inhibitory
effect of IL-1 on IL-6-induced expression of
2M, we analyzed the effect of the NF-B
inhibitor I-B on IL-6-induced 2M
promoter activation. We cotransfected HepG2 cells with I-B and an
2M promoter-reporter construct and measured
the IL-6-dependent induction of the reporter gene in the presence or
absence of IL-1. As shown in Fig. 4A, cotransfection of
I-B blocked the inhibitory effect of IL-1 on IL-6-induced
activation of the 2M promoter. Since
overexpression of I-B is known to inhibit NF-B activation,
these data indicate that activation of NF-B plays an important role
for the inhibitory effect of IL-1 on the IL-6-mediated activation of
the 2M promoter.
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B by IL-1 is
responsible for the inhibition of the IL-6-induced
2M promoter activation, we cotransfected HepG2
cells with the NF-B subunits p65 or p50 and an
2M promoter-reporter construct and measured
the IL-6-dependent induction of the reporter gene in the presence
or absence of IL-1. As shown in Fig. 4B,
cotransfection of p65 or p50 themselves already inhibits the
IL-6-induced activation of the 2M promoter.
The inhibitory effect of IL-1 on 2M
promoter activation by IL-6 experienced no further enhancement by
cotransfection with p65 or p50. These data support the idea that
activation of NF-B negatively modulates STAT3-dependent activation
of the 2M promoter.
Both NF-B binding sites within the 2M promoter
confer responsiveness of the promoter to the inhibitory activity of
IL-1, but are also essential for IL-6-mediated promoter activation
The proximal as well as the distal STAT3 binding sites within the
2M gene promoter overlap with potential
NF-B/DNA binding sites (Fig. 2A; framed vs hatched
boxes). Since we found that IL-1 exerts its inhibitory activity on
the IL-6-induced 2M gene expression through
NF-B, we analyzed whether this activity of IL-1 could be
overcome by mutating the NF-B sites within the
2M promoter fragment. Therefore, point
mutations within the proximal or distal NF-B consensus sequences
were introduced in the 2M promoter of the
reporter constructs. As shown in Fig. 2A, two
C
A substitutions at positions -155 (mpNF) and -175
(mdNF) (underlined) were generated to affect NF-B binding to the
2M promoter fragment. Minimal point mutations
were used to keep encroachment of the rest of the promoter as low as
possible and to leave the STAT3 binding sites intact. To analyze the
changes in DNA affinity of NF-B or STAT3 achieved by the mutations
introduced into the 2M promoter, competition
assays were chosen as a very reliable approach. Nuclear extracts from
IL-6-stimulated HepG2 cells were used as a source for activated STAT3.
Fig. 5A shows that a promoter
fragment bearing both mutated NF-B sites (mdNF mpNF) is as efficient
as a nonmutated DNA fragment to compete with the labeled DNA fragment
for STAT3 binding. This demonstrates that neither the CA
substitution at position -155 nor the one at -175 interferes with
STAT3 binding to the promoter. The effect of these mutations on the
binding of NF-B to the promoter fragment was analyzed in a similar
assay, but with nuclear extracts of IL-1-stimulated cells as a
source for NF-B (Fig. 5B). The nonmutated DNA fragment
was very efficient to compete with the labeled DNA fragment for NF-B
DNA binding. In contrast, a joint mutation of both NF-B binding
sites in the promoter fragment (mdNF mpNF) led to a reduced competition
indicative for a significantly reduced affinity to NF-B. The
affinity of NF-B to the promoter fragment was less affected by a
single CA point mutation at position -155 (mpNF) or
-175 (mdNF). This moderate effect of a single point mutation is
probably due to the other (remaining) intact NF-B binding site.
These data confirm that the point mutations introduced into the two
NF-B binding sites overlapping with the STAT3 sites do not influence
STAT3 binding, but interfere with NF-B binding to the
2M promoter element.
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B sites within the
2M promoter confer the promoter to respond to
the inhibitory activity of IL-1. Therefore, promoter constructs
containing the mutated proximal (-155 CA) or mutated
distal (-175 CA) NF-B/DNA-binding motif were
subjected to reporter gene assays (Fig. 5C). Mutation of the
proximal as well as the distal NF-B binding site led to an enhanced
IL-6-dependent reporter gene induction, suggesting that both sites are
functional and counteract promoter activation. However, none of these
mutations impaired the inhibitory effect of IL-1, demonstrating that
a single intact NF-B binding site within the promoter is sufficient
to exert the influence of IL-1 on 2M gene
expression. Therefore, we introduced CA substitutions
into both the proximal and the distal NF-B binding sites. Most
surprisingly, this construct displayed almost no basal or
IL-6-inducible activity, although it has been shown that point
mutations of both NF-B sites do not interfere with STAT3 binding to
the promoter (Fig. 5A). To exclude the possibility that
mutations within the proximal and distal NF-B binding sites create
new binding sites for an inhibitory factor that interferes with the
promoter inducibility, we performed gel-shift experiments with the
correspondingly mutated DNA fragment (Fig. 5D). No
additional protein/DNA complexes appeared in response to the C to A
mutations within the NF-B binding sites of the
2M promoter. Furthermore, DNA binding of STAT3
was unaffected by these mutations, as already shown in Fig. 5A. All these data provide strong evidence that the NF-B
motifs in the 2M promoter, overlapping with
the STAT3 binding sites, exert a negative regulatory function in
IL-6-induced 2M promoter activation.
Since the presence of at least one intact NF-B/DNA binding site
seems to be indispensable for proper promoter function, we next
analyzed whether NF-B binds to the STAT-responsive region of the
endogenous gene in cells that have not been stimulated with IL-1.
Therefore, we performed chromatin immunoprecipitation from primary rat
hepatocytes using an NF-B-specific Ab. The origin of the
coprecipitated endogenous DNA fragment was proven by PCR with two sets
of DNA primers corresponding to the rat 2M
gene promoter regions from -12 to -419 (Fig. 5E,
upper panel) and -103 to -283 (Fig. 5E,
lower panel). As visible in lanes 4 and
5 of Fig. 5E, NF-B binds prior to and after
stimulation with IL-6 to the endogenous 2M
promoter, supporting the idea that NF-B binding to the promoter is
essential for proper promoter function.
The inhibitory effect of IL-1 on the promoters containing a unique
mutated distal or proximal NF-B site was completely blocked by
cotransfected I-B (Fig. 6), which
is in line with the observation for the 2M
wild-type promoter (Fig. 4A). This further demonstrates that
IL-1 is able to exert its NF-B-mediated inhibitory function on
the 2M promoter through a single NF-B
binding site within the promoter.
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B binding sites in the 2M promoter act
negatively on gene induction mediated through both STAT3 binding sites
Both the proximal as well as the distal NF-B binding site
confer responsiveness of the 2M promoter to
the inhibitory activity of IL-1 through NF-B (Figs. 5C
and 6). We next analyzed whether the NF-B binding sites affect gene
induction mediated through the individual STAT3 binding sites (Fig. 7, C and D).
Therefore, promoter constructs were generated containing a single
functional STAT3 binding site in combination with a single functional
NF-B binding site (Fig. 2A). The mutations within the
STAT3 response elements were verified not to inhibit NF-B binding
(Fig. 7A), but to inhibit STAT3 DNA binding (Fig. 7B). The promoter fragment with both STAT3 binding sites
mutated (mdST mpST) competed for NF-B binding with the labeled
NF-B probe as efficient as the wild-type 2M
promoter fragment (wt; Fig. 7A), demonstrating that the
mutation does not interfere with NF-B DNA binding. In contrast, the
mdST/mpST-mutated oligonucleotide did not compete for STAT3 binding to
the labeled promoter fragment (Fig. 7B; right
panel), confirming that this oligonucleotide does not contain any
functional STAT3 binding site. Both DNA fragments containing a single
mutated STAT3- and NF-B-binding motif ((mdNF mpST) and (mdST mpNF))
competed for STAT3 DNA binding with the labeled promoter fragment,
demonstrating that both fragments are still capable of binding STAT3.
The (mdNF mpST) oligonucleotide was found to be less efficient for
competition than the (mdST mpNF) oligonucleotide. This is in line with
the described observation that STAT3 binds with higher affinity to the
proximal than to the distal STAT3-responsive element of the
2M promoter (18).
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B binding sites described above into promoter-reporter
constructs. Similar to the sole disruption of the proximal NF-B site
(Fig. 5C), combined mutations of the proximal NF-B
binding site (CA -155) and the distal STAT3 binding site
(TC -187) strongly enhanced the promoter response to
IL-6 when compared with the wild-type 2M
promoter. The IL-6-induced activation of this construct was still
inhibited by IL-1. Moreover, mutation of the proximal STAT3 binding
site (TC -165) and the proximal NF-B binding site
(CA -155) led to a largely diminished promoter response
to IL-6 stimulation (Fig. 7C). Nevertheless, this response
was also inhibited by preincubation with IL-1, suggesting that the
distal NF-B site displays its inhibitory function on the
IL-6-induced promoter on both STAT3-responsive elements.
A similar observation was made for the NF-B binding
site overlapping with the proximal STAT3 binding site. Neither the
combined mutation of the distal NF-B (CA -175) site
with the distal STAT3 binding site (TC -187) nor
disruption of the distal NF-B site plus the proximal STAT3
(TC -165) site led to an IL-1-insensitive promoter
(Fig. 7D). The observation that the
2M promoter is much more sensitive to a
mutation in the proximal than in the distal STAT3-responsive element
further demonstrates the importance of the proximal STAT binding site
and confirms previous findings (18).
Down-regulation of other IL-6-inducible genes by IL-1
Tandem STAT3 binding sites are not unique for the
2M promoter, but are also found in the
regulatory elements of other IL-6-inducible genes such as the
1ACT and the SOCS3 gene promoter. Within these
promoter regions, similar to the 2M gene
promoter, putative NF-B binding sites overlap with STAT3 binding
sites (Fig. 2B). Thus, it is well possible that the
investigated regulatory mechanism concerning the
2M promoter is of more general importance,
i.e., the 1ACT and the SOCS3 genes are
similarly modulated as the 2M gene. The
following experiments were conducted to give support to this idea.
As shown in Fig. 8A, a
reporter construct containing a promoter fragment (-379 to +25) of the
1ACT gene could be activated by stimulation of
transfected HepG2 cells with IL-6, but was also negatively affected by
costimulation with IL-1. As found for the
2M gene promoter, IL-1 is suggested to
exert its inhibitory activity on the analyzed part of the
1ACT promoter also through NF-B since
expression of I-B at least partially blocked its inhibitory
effect.
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(Fig. 8B). As shown in Fig. 8B, stimulation with IL-6
led to strong and transiently increased SOCS3-mRNA levels within 1
h. Preincubation with IL-1 for 10 min attenuated this increase in
SOCS3-mRNA levels upon stimulation with IL-6. Again, we analyzed
whether IL-1 exerts its inhibitory activity on the IL-6-dependent
increase in SOCS3-mRNA amounts through NF-B (Fig. 8C).
Indeed, the effect of IL-1 was partially reduced in cells treated
with a cell-permeable peptide (SN50, Fig. 8C, lane
4), which carries a functional cargo representing the nuclear
localization sequence of the NF-B subunit p50 to impede nuclear
translocation of endogenous p50. In contrast, a control peptide (SN50
M), having mutations in two of ten residues within the nuclear
localization sequence, did not affect the inhibitory activity of
IL-1 (Fig. 8C, lane 5). However, since SN50
only interferes with nuclear translocation of the p50 subunit, and not
the p65 subunit, of NF-B, these experiments might not reflect the
total extent of the contribution of NF-B toward inhibition of
IL-6-induced SOCS3-mRNA expression by IL-1 (22).
In conclusion, these data lead us to the speculation that the dual
function of NF-B as a positive and negative regulator of gene
expression, described in this study in more detail for the
2M promoter, might also be true for other
promoters such as those of the 1ACT and the
SOCS3 genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is one of the major events following the
onset of an inflammatory response mainly initiated by proinflammatory
cytokines such as IL-1 and TNF-. On the other hand, activation of
STAT factors is an important part of the action of cytokines and IFNs
released during the inflammatory response (1). In the
past, it became increasingly evident that there exists an extensive and
complex cross-talk between various signal transduction cascades
initiated by simultaneously acting cytokines, largely modulating
cellular responses toward a single stimulus.
For example, it is well documented that IL-1, TNF-, as well as
LPS counteract IL-6 signal transduction and IL-6-mediated gene
induction (19, 20, 21). Several mechanisms for the negative
regulation of IL-6 signal transduction have been identified: 1) The
protein tyrosine phosphatase SH2-containing protein tyrosine
phosphatase 2 is activated by IL-6 and counteracts initiation of IL-6
signaling (28, 32, 33), although the direct molecular
targets of SH2-containing protein tyrosine phosphatase 2 still have to
be determined. 2) Protein inhibitors of activated STATs have been
cloned and found to bind and inhibit tyrosine-phosphorylated STAT
transcription factors (34, 35). 3) SOCS proteins have been
recognized as IL-6-induced feedback inhibitors of the Jak kinases
(36, 37, 38). 4) Nuclear tyrosine phosphatases have been shown
to inactivate STATs (39). 5) The proteasome has been found
to degrade STAT factors (40).
Very recently, data have been presented suggesting that TNF- and LPS
act as inhibitors of IL-6 signaling through the induction of SOCS3
(19, 20). Analyzing the mechanism underlying the
IL-1-mediated attenuation of IL-6 signal transduction, it was
observed that IL-1, in contrast to TNF- and LPS
(19), did not induce SOCS3 gene expression (visible in
Fig. 8, B and C) and hardly affected
IL-6-mediated STAT3 activation (Fig. 1, upper panel). In
this study, we identified NF-B as a mediator of IL-1-dependent
negative regulation of IL-6-inducible genes (Figs. 4, 5C, 6,
7C, 7D, and 8). NF-B exerts its negative
regulatory function on the 2M promoter by
counteracting DNA binding of STAT3 at overlapping STAT3/NF-B binding
sites (Figs. 3; 5, A, B, D, and
E; and 7, A and B). We propose that
this activity is responsible for the inhibitory effect of IL-1 on
IL-6-induced 2M promoter activation.
Surprisingly, although NF-B acts as a competitive inhibitor for
STAT3 DNA binding, at least one intact NF-B consensus site was found
to be crucial for the STAT3-dependent activation of the
2M gene promoter (Fig. 5C).
Furthermore, additional data are provided, suggesting that the
activation of NF-B by IL-1 also inhibits the induction of an
1ACT reporter gene construct (Fig. 8A) as well as of the SOCS3 gene (Fig. 8, B and
C). Thus, competition between NF-B and STAT3 seems to be
of more general relevance for inhibition of IL-6-induced gene induction
by IL-1.
Cross-talk between STAT factors and NF-B has also been
described for other promoters, for example, the one of the IFN
regulatory factor-1 gene. This gene promoter was shown tobe
synergistically activated by TNF- and IFN- (41, 42, 43).
The synergism was suggested to be due to the independent
interaction of the involved transcription factors with components of
the basal transcription machinery. Prolactin-activated STAT5B inhibits
NF-B-dependent IFN regulatory factor-1 promoter activation by
squelching limited coactivators (44). These modes of
cross-talk between STAT factors and NF-B are in contrast to our
observations for the 2M promoter, in which
NF-B and STAT3 compete for overlapping DNA binding sites. More
similar to the conditions described for the 2M
promoter, in this work is the regulation of the E-selectin gene
promoter by STAT6 and NF-B. At this promoter, IL-4-induced STAT6
binding counteracts TNF--induced expression of the E-selectin gene
via competition with NF-B for DNA binding at overlapping
STAT6/NF-B binding sites (45). Thus, in this case, the
roles for STAT and NF-B are reversed when compared with the
2M promoter.
As shown in this study, at least one nonmutated NF-B consensus site
is crucial for promoter activation. 2M
promoter constructs with both NF-B binding sites mutated displayed
almost no basal or IL-6-inducible activity (Fig. 5C). Thus,
it might be that activated NF-B at low concentrations, not high
enough to counteract STAT3 DNA binding, further represents an important
constituent of the transcription machinery involved in STAT3-mediated
transcriptional activation. In this respect, it is important to note
that the NF-B subunit p65 has been shown to specifically engage
CBP/p300 for maximal transcriptional stimulation of the IL-6 gene
promoter by its histone acetyltransferase activity (46).
Indeed, we were able to show NF-B binding to the endogenous
2M promoter in both unstimulated and
IL-6-treated primary rat hepatocytes (Fig. 5E). We did not
detect direct NF-B binding to DNA in nuclear extracts from cells
stimulated with IL-6 alone in EMSA (Fig. 3A). This suggests
that under conditions that allow STAT3-dependent gene induction,
NF-B has no high affinity to the NF-B binding site of the
promoter element. However, DNA binding of NF-B became visible after
elimination of STAT3 DNA binding by interfering STAT3 Abs, indicating
that NF-B is also present in nuclear extracts from cells stimulated
with IL-6 alone (Fig. 3A). Since removal of STAT3 obviously
enables NF-B to bind the DNA in the gel retardation assay, these
data emphasize the idea that the ratio of STAT3 and NF-B is critical
for the affinity of these transcription factors to bind the
2M promoter element. This idea is further
supported by the observation that increased amounts of activated
NF-B after IL-1 stimulation led to a loss of STAT3 DNA binding
(Figs. 1 and 3). Moreover, prevention of the IL-1-induced NF-B
activation blocks the inhibitory effect of IL-1 on STAT3-dependent
IL-6-mediated activation of the 2M promoter
(Fig. 4A), an effect that is conferred by each of both
NF-B motifs (Fig. 6). In line with these data is the observation
that expression of the NF-B subunits p65 or p50 mimics
IL-1-dependent signal attenuation (Fig. 4B).
Another possibility to explain the IL-1-mediated repression of the
2M promoter would be the specific induction of
promoter binding by negative regulatory p50 homodimers
(47) in IL-1-stimulated cells. Such a putative
IL-1-mediated switch from p50/p65 to p50/p50 promoter binding is
considered to be unlikely since p65 has been demonstrated to be present
within the NF-B/DNA complex induced by IL-1 (Fig. 3A)
and, again, its overexpression of p65 alone mimics the inhibitory
effect of IL-1 (Fig. 4B).
Further preliminary evidence for a competition of STAT3 and NF-B for
binding the 2M promoter was given by Zhang and
Fuller (26). However, these authors concentrated on the
isolated proximal STAT3 binding site and did not analyze a larger part
of the 2M promoter containing the complete
tandem motif. Furthermore, this observation was not followed up with
respect to its functional implications for inhibition of IL-6-induced
gene expression by IL-1, LPS, or TNF-.
In our study, we analyzed an extended part of the
2M promoter containing both STAT binding
sites. The arrangement of tandem STAT sites has been shown in many
promoters to mediate binding of tandem STAT dimers, which is
strengthened by association through the N-terminal domain of the STATs
(8). This appears to be important at promoters in which
one site does not fit the consensus sequence. The
2M promoter meets these conditions. As shown
in Fig. 7, C and D, the proximal STAT3 site,
which fits the STAT consensus better than the distal STAT site, is much
more potent to mediate IL-6-dependent promoter activation than the
distal site. Furthermore, both sites act somehow synergistically on the
promoter. Thus, the relevance of individual binding sites should be
analyzed in context of the other binding sites within the
2M promoter.
In general, the loss of promoter activity of mutants lacking both
NF-B consensus sequences (Fig. 5C) might be due to the
introduction of new binding sites for unidentified inhibitory proteins
within the mutated promoter element. However, we exclude this
possibility since mutation of a single NF-B site did not reduce, but
rather increased promoter activity (Figs. 5C, and 7,
C and D). Furthermore, no additional protein/DNA
complexes were observed in EMSA with the corresponding DNA element
(Fig. 5D).
Finally, one could speculate that there is another, unidentified
protein binding to the NF-B site that is responsible for working
synergistically with STAT3 to activate transcription. The unspecific
band visible in all EMSA performed with the 2M
promoter fragment could represent a candidate protein. Thus, mutation
of both NF-B consensus sequences should eliminate DNA binding of
this protein. This explanation seems also to be unlikely since the
mutations introduced into the promoter fragment did not affect DNA
binding of this protein (Fig. 5D).
In summary, activation of NF-B is shown to be a crucial negative
regulatory step by which the proinflammatory cytokine IL-1 controls
STAT3-dependent gene induction by IL-6. Considering the fact that IL-6
displays distinct antiinflammatory properties, it is attractive to
speculate that proinflammatory mediators such as LPS, TNF-, or IL-1
down-regulate IL-6 signaling to enforce the inflammatory response.
Being aware that antiinflammatory activities have been described for
several APP induced by IL-6 (48, 49, 50), this hypothesis
becomes even more conclusive.
|
Acknowledgments |
|---|
B expression vector, p65 expression vector, and the SOCS3
cDNA were kindly provided by K. Brand (Munich, Germany), M.
Nourbakhsh and H. Hauser (Braunschweig, Germany), and D.
Hilton (Parkville, Australia), respectively. We gratefully acknowledge
the generous supply of human rIL-1 from D. Boraschi (L’Aquila,
Italia). We thank W. Frisch and M. Ruhl for technical
assistance. |
Footnotes |
|---|
2 Current address: Klinik für Gastroenterologie, Hepatologie und Infektiologie, Medizinische Klinik der Heinrich-Heine Universität, 40255 Düsseldorf, Germany.
3 Address correspondence and reprint requests to Dr. Peter C. Heinrich, Institut für Biochemie, Klinikum der RWTH Aachen, Pauwelsstrae 30, D-52074 Aachen, Germany. E-mail address: heinrich{at}rwth-aachen.de
4 Abbreviations used in this paper: APP, acute-phase protein; 1ACT, 1-antichymotrypsin; 2M, 2-macroglobulin; Jak, Janus kinase; SOCS, suppressor of cytokine signaling.
Received for publication November 15, 2000. Accepted for publication May 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and tumor necrosis factor on the mRNA induction of acute-phase proteins. Eur. J. Immunol. 18:739.[Medline]
receptor components. Science 263:92.B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma. J. Biol. Chem. 270:31129.2-macroglobulin gene. Nucleic Acids Res. 17:1121. induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages. FEBS Lett. 463:365.[Medline]
. J. Immunol. 163:2640.B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270:14255.B on an overlapping DNA binding site. Biochem. Biophys. Res. Commun. 237:90.[Medline]
B/IB story. Adv. Cancer Res. 66:255.[Medline]
-activated STAT1 by the ubiquitin-proteasome pathway. Science 273:1717. and tumor necrosis factor- in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor B. J. Biol. Chem. 272:14899. and IFN signalling pathways through synergistic induction of IRF-1/ISGF-2 is mediated by a composite GAS/B promoter element. Nucleic Acids Res. 25:4346.B-mediated signaling. Mol. Endocrinol. 14:114.-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-B. J. Biol. Chem. 272:10212.B engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J. Biol. Chem. 274:32091.2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-1 in mice. J. Clin. Invest. 87:39.
2-macroglobulin as a cytokine-binding plasma protein: a study with rh-interleukin-1 and rh-interleukin-6. Ann. NY Acad. Sci. 737:439.[Medline]
synthesis by human peripheral blood mononuclear cells. J. Exp. Med. 178:1629.This article has been cited by other articles:
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