From the Department of Gene Regulation and
Differentiation, GBF-National Research Institute for Biotechnology,
Mascheroder Weg 1, D-38124 Braunschweig and § Institute of
Pharmacology, Medical School Hannover, Carl-Neuberg-Strasse 1,
D-30625 Hannover, Germany
Received for publication, August 18, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Interleukin (IL)-8, a prototypic chemokine, is
rapidly induced by the pro-inflammatory cytokine IL-1 but is barely
detectable in noninduced cells. Although there is clear evidence that
the transcription factor NF- A hallmark of inflammation is the invasion of activated leukocytes
into the injured tissue. This step is critically dependent on the rapid
expression of chemokines, cytokines whose main function is to attract
and activate leukocytes at sites of infection or damage (1).
IL1-8 was the first
identified member of the still growing chemokine family and represents
the prototype human chemokine (2). IL-8 synthesis, low or undetectable
in normal noninflamed tissue, can be induced in vivo as well
as in a wide variety of cells in vitro by pro-inflammatory
cytokines such as IL-1 or tumor necrosis factor (3, 4) or as a direct
consequence of contact with pathogens like bacteria (5, 6), viruses (7,
8). and cell-stressing agents (9-12). The regulation of IL-8 by IL-1
is of particular pathophysiological importance, because blockade of
IL-1 receptors by application of the IL-1 receptor antagonist consistently reduces tissue neutrophilia in a variety of disease models
presumably by preventing IL-1-induced synthesis of IL-8 and related
chemokines (13).
As chemokine gene expression is tightly regulated, understanding the
underlying molecular basis of this control is likely to yield novel
insight into the pathology of inflammation and may result, ultimately,
in development of novel anti-inflammatory drugs.
In that respect the dimeric transcription factor NF- On the other hand, proteins have been described that repress or inhibit
NF- Previous studies have shown that a sequence spanning the nucleotides
In a search for potential repressory mechanisms of IL-8 transcription,
we found a sequence within the IL-8 promoter that showed high homology
with a regulatory element of the IFN- Here we report a second gene, interleukin-8, whose basal expression is
controlled by NRF. Furthermore, we provide evidence that by binding to
the NRE of the IL-8 promoter, NRF plays an additional role and acts as
a coactivator of IL-1-induced IL-8 gene expression.
Cells and Materials--
HeLa cells stably transfected with the
plasmid pUHD15-1 expressing the tet transactivator
protein were obtained from H. Bujard and used throughout this study
(39). Cell lines were cultured in Dulbecco's modified Eagle's medium
complemented with 10% fetal calf serum. E64
(trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), pepstatin, leupeptin, phenylmethanesulfonyl fluoride (PMSF), and all
other chemicals were from Sigma; [ Plasmids--
The expression plasmid for GST-JUN (amino acids
1-135) was a kind gift of J. R. Woodgett. GST fusion proteins
were expressed and purified from Escherichia coli by
standard methods. Plasmid pMCL-HAMKK1R4F, encoding
constitutively active MKK1 ( Transfections and Preparation of Cell Extracts--
Transient
transfections were performed by the calcium phosphate method exactly as
described (41). For determination of reporter gene activity, cells were
lysed in ice-cold potassium phosphate buffer (100 mM, pH
7.8), containing 0.2% Triton X-100, 1 µg/ml pepstatin, 2,5 µg/ml
leupeptin, and 1 mM PMSF. After 15 min on ice, lysates were
cleared by centrifugation at 10,000 × g, and Immunoprecipitation and JNK Assay--
Cell extract protein
(100-250 µg) from cells transfected with indicated plasmids was
diluted in 500 µl of immunoprecipitation (IP) buffer (20 mM Tris, pH 7.4, 154 mM NaCl, 50 mM
NaF, 1 mM Na3VO4, 1% Triton
X-100). Samples were incubated for 3 h with 2 µl of antibody
SAK9 to which 20 µl of protein A-Sepharose was added subsequently.
Beads were spun down, washed 3× in 1 ml of IP buffer, and resuspended
in 10 µl of IP buffer. Then 10 µl of GST-JUN (1 µg) and 10 µl
of kinase buffer (150 mM Tris, pH 7.4, 30 mM
MgCl2, 60 µM ATP, 4 µCi of
[ Electrophoretic Mobility Shift Assay (EMSA)--
Double-stranded
oligonucleotides corresponding to the IL-8 promoter sequences shown in
Fig. 1 were end-labeled using [ Inducible Expression of NRF mRNAs--
HeLa tTA cells were
transfected with 5 µg of pTBC, pTBC-NRF, or pTBC Northern Blot Analysis--
Northern blots were performed as
described (38, 42). cDNA probes against NRF, neomycin
phosphotransferase, and interleukin-8 were generated by polymerase
chain reaction.
Western Blots--
Western blots were performed as described
(41). The endogenous NRF protein was detected by immunoprecipitation
using an equal mix of polyclonal antibodies directed against aa
256-272 and 272-288. Following immunoprecipitation, cleared extracts
were analyzed by Western blot using a mix of polyclonal antibodies directed against aa 25-45, 175-191, and 364-382 of NRF. This
procedure has been described in detail elsewhere (38).
Enzyme-linked Immunosorbent Assay--
IL-8 protein
concentrations in the cell culture medium were determined using the
human IL-8 duo set kit (R & D Systems) exactly following the
manufacturer's instructions.
The NRE cis-Element Is Required for Basal Repression and
IL-1-induced Activation of the IL-8 Promoter--
Although the IL-8
and IFN-
To analyze further the requirement of the NRE site for inducible IL-8
transcription, we tested if any of the known signaling pathways
triggered by IL-1 target the NRE. IL-1 induces
NF-
A significant activation of the IL-8 promoter, although lower than with
MEKK1 and TAK1, was observed by selective activation of the ERK or p38
MAPK pathways. This was achieved by transfecting the constitutively
active forms of MKK6 and MKK1, MKK62E (41, 45, 46), and
MKK1R4F (47), respectively. However, this induction of the
IL-8 promoter was not affected by the NRE mutation (Fig. 2B). Thus ERK and p38 MAPK pathways are unlikely to
contribute to IL-1-, MEKK1-, and TAK1-mediated
NRE-dependent IL-8 transcription.
These results suggest that the NRE is also required for transcriptional
induction by stimuli that activate NF-
Since the NRE in the IFN- The NRE Is Not Required for NF-
We investigated the formation of IL-1-inducible complexes between
proteins and IL-8 promoter DNA by EMSA. As shown in Fig. 3, nuclear extracts from HeLa cells
treated for 30 min with IL-1 formed two complexes (I and II), which
were absent in untreated cells. The appearance of two IL-1-inducible
complexes binding to the IL-8 promoter is consistent with the results
of others (36, 49, 50). Both complexes contained p65 NF-
In the presence of an NRE mutant oligonucleotide, both IL-1-inducible
complexes I and II were formed and were supershifted with the anti-p65
antibodies, indicating that p65 NF-
In the presence of NRF antiserum, but not preimmune serum, a slower
migrating complex was detected in extracts from IL-1-stimulated and
untreated cells. This complex failed to form with the NRE mutant
oligonucleotide. Furthermore, a prominent constitutive protein-DNA
complex (labeled III) formed with the wild type IL-8 promoter
oligonucleotide, but like the complexes with anti-NRF antibodies, it
could not be detected with the NRE mutant oligonucleotide. Although we
did not observe a classical supershift with the anti-NRF antibodies,
these data suggest that NRF may be part of the constitutive protein-DNA
complex III whose formation depends on an intact NRE (Fig. 3).
Therefore, in nuclear extracts of unstimulated as well as of
IL-1-stimulated cells, NRF binding to the IL-8 promoter requires an
intact NRE site, at least in vitro. These data also suggest that the reduction of IL-1-induced IL-8 promoter activity observed for
the NRE mutation (Fig. 2, A and B) is not caused
by an altered DNA binding and transactivation of the promoter by p65
NF- Activation of the IL-8 Promoter by p65 NF-
IL-1, MEKK1, and TAK1 activate NF-
A constitutively active mutant of MKK7, MKK73E, in which
the regulatory amino acids Ser-271, Thr-275, and Ser-277 were replaced by glutamic acid (41, 58) was expressed together with JNK2 and p65
NF-
Like p65 NF- NRF Is Required for Constitutive Repression and for IL-1-induced
Expression of the Endogenous IL-8 Gene--
Based on these results, we
performed experiments to confirm that NRF also plays a role in
regulation of endogenous IL-8 gene expression. HeLa cells were stably
transfected with the sense or a 300-base pair antisense NRF cDNA
under the control of a tetracycline-sensitive promoter (38). The
induction of antisense RNA resulted in efficient reduction of nuclear
NRF as determined by Western blot analysis of immunoprecipitated
endogenous NRF (Fig. 6A). The
expression of endogenous IL-8 and ectopically expressed sense and
antisense NRF mRNAs was simultaneously monitored by Northern blot
analysis. No IL-8 mRNA was detected in the presence of
tetracycline. Omitting tetracycline led to a significant expression of
the endogenous IL-8 mRNA in the NRF antisense mRNA expressing
cells (Fig. 6B). In contrast, in cells expressing the
full-length NRF sense mRNA, or in cells transfected with empty
vector, no endogenous IL-8 mRNA expression was detectable upon
withdrawal of tetracycline.
As expected from the IL-8 mRNA data, induction of the antisense NRF
mRNA fragment also increased basal IL-8 protein levels by at least
2-fold (Fig. 6C). These results indicate that reducing NRF
protein expression derepresses the interleukin-8 gene. In analogy to
the IFN-
The experiments with the NRE-mutated IL-8 promoter suggested that NRF,
in addition to its repressory role, functions as a coactivator of IL-8
gene expression in IL-1-stimulated cells. IL-1 treatment strongly
induced IL-8 secretion in HeLa cells (Fig. 6C). This
IL-1-induced IL-8 secretion was inhibited by more than 3-fold by
expressing NRF antisense RNA. This result confirms that NRF is required
for maximal IL-8 gene expression.
The expression of NRF sense mRNA resulted in a 25% higher
IL-1-induced IL-8 secretion as in cells transfected with empty vector. This is in agreement with the data presented in Fig. 5B,
showing that NRF cotransfection enhances NF-
Taken together the experiments shown in Fig. 5 and Fig. 6 led us to
conclude that NRF abundance is critical for IL-8 repression and as well
provides strong evidence for a role of NRF as a cofactor in
IL-1-induced IL-8 transcription.
Repressory mechanisms may play an important role in the control of
chemokine expression. Here, we describe the role of NRF and its
DNA-binding element NRE in the regulation of both repression and
activation of the IL-8 promoter.
NRF was found as a suppressor of basal IFN- A competitive mechanism for repression of the IL-8 gene has been
described earlier. Wu et al. (36) showed that the
transcription factor Oct-1 binds to the C/EBP site in vitro.
Converting the C/EBP-binding site to a C/EBP consensus site abolished
Oct-1 binding in vitro and derepressed IL-8 transcription in
reporter gene assays. In their model, transcription of the IL-8
promoter is induced by replacing Oct-1 with NF- Most interesting, several lines of evidence indicate that NRF is also
required for a strong activation of the IL-8 gene by IL-1. First, a
mutant NRE sequence leads to a substantially reduced IL-1-induced IL-8
transcription (Fig. 2). Second, binding of NRF to the NRE is abolished
by a mutation in the NRE (Fig. 3). Third, expression of antisense
mRNA of NRF impairs IL-1-induced IL-8 secretion (Fig. 6).
The mechanism by which NRF contributes to IL-1-induced IL-8 promoter
activation remains elusive. NF- Induction of IFN- Several other transcriptional regulators have been described that are
able to act as activators and repressors. The molecular basis of these
dual roles is diverse (64). Our results show that NRF represents a
novel type of repressor molecule that is able to switch to an
activator. This mechanism depends on the type of inducer and the
regulation and structure of the transcriptional complex. Our
description of a previously undetected mechanism of IL-8 regulation
adds to our understanding of the complexity of this chemokine
expression. This type of regulation might also be relevant for other
genes involved in inflammatory events. The combination of NF-B plays a central role in inducible IL-8 transcription, very little is known about the cis-elements
and trans-acting factors involved in silencing of the IL-8
promoter. By sequence comparison with the interferon-
promoter, we
found a negative regulatory element (NRE) in the IL-8 promoter
overlapping partially with the NF-
B response element. Here we show
that an NF-
B-repressing factor (NRF) binds to the IL-8 promoter
NF-
B-NRE. Reduction of cellular NRF by expressing NRF antisense RNA
results in spontaneous IL-8 gene expression. In contrast, IL-1-induced IL-8 secretion is strongly impaired by expressing NRF antisense RNA.
Mutation of the NRE site results in loss of NRF binding and increased
basal IL-8 transcription. On the other hand IL-1-induced IL-8
transcription is decreased by mutating the NRE. These data provide
evidence for a dual role of the NRF in IL-8 transcription. Although in
the absence of stimulation it is involved in transcriptional silencing,
in IL-1-induced cells it is required for full induction of the IL-8 promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B has emerged as
a key molecule in the transcriptional regulation of genes relevant to
inflammation (14), including chemokines such as IL-8 (15). The activity
of NF-
B is controlled at multiple levels. First, extracellular
signals activate the recently identified inhibitor of NF-
B kinase
complex that induces phosphorylation-dependent proteolytic
degradation of I
B proteins and allows translocation of
NF-
B from the cytosol to the nucleus (16). Second, transcriptional activity of NF-
B is potentiated by inducible phosphorylation (17-23), cooperation with AP-1 (24), by binding to the CREB-binding protein/p300 coactivator (25) and integration of NF-
B into multiprotein transcriptional complexes such as the IFN-
enhanceosome (26, 27).
B activity. Recombination signal sequence-binding protein J
(RBP-J
) constitutively binds to NF-
B sites in the IL-6 and
NF-
B 2 (p100/p52) promoters and represses basal transcription (28-30). Upon induction RBP is displaced by NF-
B, relieving
transcriptional repression. Nuclear hormone receptors physically
interact with NF-
B in the absence of DNA (31) and thereby interfere
with inducible IL-6 (32) or IL-8 transcription (33). The zinc finger protein A20 represses tumor necrosis factor-induced
NF-
B-dependent IL-6 and granulocyte
macrophage-colony stimulating factor (GMCSF) gene expression by a
cytosolic pathway (34).
1 to
133 within the 5'-flanking region of the IL-8 gene is
essential and sufficient for transcriptional induction of the gene by
most stimuli including IL-1 (15). This region contains a single NF-
B
element that is required for activation in all cell types studied, as
well as an AP-1 and a C/EBP site (15, 35). In the absence of immune
stimulation, this promoter fragment is transcriptionally silent (15,
36). This suggests that it may be under negative transcriptional control.
promoter. This 16-base pair
sequence spans the decameric binding site for NF-
B which overlaps
with a previously characterized negative regulatory
cis-element (NRE). The NRE is required for
position-independent silencing of the NF-
B site of the IFN-
promoter (37). A protein called NF-
B-repressing factor (NRF) was
cloned recently which specifically binds to this site (38). NRF
represses basal IFN-
transcription but, although permanently bound
to the promoter, is not required for virus-induced IFN-
gene
expression (38).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was
purchased from Hartmann Analytics. Rabbit antiserum SAK9 to the C
terminus of JNK2 (40) was a kind gift of J. Saklatvala. Antibodies
directed against NRF were raised in rabbits immunized against five
different peptides based on the antigenic index of the NRF protein
sequence (aa 25-45, 175-191, 256-272, 272-288, and 364-382).
Antibodies 12CA5 directed against hemagglutinin (HA) and 9E10 against
c-MYC epitopes were from Roche Molecular Biochemicals.
Antibodies against NF-
B p65 (sc-372) were from Santa Cruz
Biotechnology. Horseradish peroxidase-coupled secondary antibodies
against mouse and rabbit IgG were from Sigma. Protein A-, G-, and
GSH-Sepharose were from Amersham Pharmacia Biotech. Human recombinant
IL-1-
a was produced as described (40).
N3/S218E/S222D), was a kind gift of
N. G. Ahn. Plasmids pFC-MEKK1 encoding amino acids 360-672 of
MEKK1 and pSV-
-galactosidase were from Stratagene and Promega,
respectively. PcDL-SR
-HATAK1 and pEFTAB1 were from E. Nishida and J. Ninomiya-Tsuji, respectively. Plasmids pCS3MT-MKK73E (Ser-271, Thr-275, and Ser-277 mutated to Glu),
peVHA-MKK62E (Ser-207 and Thr-211 mutated to Glu),
peVHA-JNK2, and the IL-8 promoter-driven luciferase reporter plasmid
pUHC13-3-IL-8pr (nucleotides 1348-1527 of the IL-8 gene) have been
described (41). Expression plasmids p65 NF-
B, pNRFVP16, pDBDVP16,
pNRF, and pGFPNRF have been described (38). PCS3MT-NRF encodes for
N-terminally MYC-tagged NRF. The tet-off expression plasmids
pTBC-NRF and pTBC-
NRFas contained the full-length NRF coding region
or nucleotides 1-300 of the coding region in antisense orientation,
respectively. Site-directed mutagenesis of NRE and NF-
B sites was
performed using the oligonucleotides described in Fig. 1. The AP-1
mutant of the IL-8 promoter plasmid has been described elsewhere (41).
All mutations described above were introduced using the Quick
ChangeTM site-directed mutagenesis kit (Stratagene). Primer
sequences used for polymerase chain reaction are available upon
request. Sequences were confirmed by automated DNA sequencing on an ABI 310 sequencer (Applied Biosystems).
-galactosidase activity was measured with reagents from
CLONTECH. Luciferase activity was determined using
reagents from Promega and normalized for
-galactosidase activity.
Nuclear and cytosolic extracts were prepared as described previously
(41). Briefly, cells were suspended and pelleted in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.3 mM Na3VO4,
20 mM
-glycerophosphate and freshly added 2.5 µg/ml leupeptin, 10 µM E64, 300 µM
PMSF, 1.0 µg/ml pepstatin, 5 mM DTT, 400 nM
okadaic acid). The pellet was resuspended in buffer A + 0.1% Nonidet
P-40 and left on ice for 10 min and then vortexed. After centrifugation
at 10,000 × g for 5 min at 4 °C, supernatants were
taken as cytosolic extracts. Pellets were resuspended in buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25%
glycerol, 0.3 mM Na3VO4, 20 mM
-glycerophosphate, 2.5 µg/ml leupeptin, 10 µM E64, 300 µM PMSF, 1.0 µg/ml pepstatin,
5 mM DTT, 400 nM okadaic acid). After 1 h
on ice nuclear extracts were vortexed and cleared at 10,000 × g for 5 min at 4 °C and supernatants collected. Protein concentration of cell extracts was determined by the method of Bradford
and samples stored at
80 °C.
-32P]ATP) were added. After 30 min at room
temperature, assays were stopped and proteins eluted from the beads by
boiling for 5 min in SDS-PAGE sample buffer(8% SDS, 100 mM
Tris, pH 6.8, 4%
-mercaptoethanol, 24% glycerol, 0.02% bromphenol
blue). After centrifugation at 10,000 × g for 5 min,
supernatants were separated on 10% SDS-PAGE. Phosphorylated proteins
were visualized by autoradiography.
-32P]ATP and T4
polynucleotide kinase and purified by gel filtration on S-200 spin
columns (Amersham Pharmacia Biotech). Protein-DNA binding reactions
were performed with 5-20 µg of nuclear extract protein, labeled
oligonucleotide, 1 µg of poly(dI-dC) in 10 mM Tris, pH
7.5, 10 mM EDTA, 0.05% (w/v) dried low-fat milk, 50 mM NaCl, 10 mM DTT, and 10% glycerol in a
total volume of 10 µl. After incubation at room temperature for 30 min, protein-DNA complexes were resolved on 5% PAGE and visualized by
autoradiography. For NRF supershifts the five
-NRF antisera were
mixed, and 1-2 µl of this mixture or of the corresponding preimmune
sera were added at the beginning of the binding reaction.
NRFas, 10 µg of
high molecular weight DNA, and 0.5 µg of pSV2PAC encoding the
puromycin acetyltransferase gene as described (38). Stably transfected
cells were selected and maintained in 4.2 µg/ml puromycin and 500 µg/ml G418 in the presence of 2 µg/ml tetracycline. More than 100 clones were pooled and tested for IL-8 and NRF expression. Tetracycline
was removed for 48 h to induce NRF sense or antisense mRNA.
Cells were then stimulated where indicated with 24 ng/ml IL-1
or
with Sendai virus (10 plaque-forming units/ml) or left untreated. After
18 h, the supernatants of HeLa-tTA cells were tested for
IL-8 expression or cells were harvested and subjected to
immunoprecipitation experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoters differ in most of their known positive regulatory
elements, sequence comparison revealed one highly homologous region
(Fig. 1). Therefore, we carried out experiments to examine a putative role of the NRE and NRF in IL-8 promoter regulation. Initially, we explored the role of the NRE in
basal and IL-1-induced regulation of IL-8 transcription using a
luciferase reporter gene under the control of a 180-base pair fragment
of the IL-8 promoter. Parallel experiments were carried out with wild
type (wt) promoter or versions mutated in the NF-
B and NRE sites
(Fig. 1). The NRE mutant IL-8 promoter contained two point mutations
immediately adjacent to the NF-
B site, which were previously found
to inactivate the NRE (37). Similarly, three distinct mutations within
the NF-
B-binding site were introduced as reported by others (36) to
create an inactive NF-
B-binding site. The wt IL-8 promoter displays
a low transcriptional activity, which is somewhat lower when the
NF-
B-binding site is mutated. Mutation of the NRE leads to a low but
significant activation of the reporter (Fig.
2A). This is consistent with
the role of the NRE in repression of basal transcription as previously
observed for the IFN-
gene (37). IL-1 treatment of cells transfected with the wild type IL-8 promoter resulted in more than 80-fold induction of promoter activity. This was strongly reduced by mutating the NF-
B site. Surprisingly, mutating the NRE also resulted in a
more than 3-fold reduction of IL-1-stimulated promoter activity (Fig.
2A).
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Fig. 1.
Sequence comparison of the
IFN- and the IL-8 promoter. Parts of the
IFN-
and IL-8 promoter proximal regulatory regions are illustrated.
Identified binding sites for transcription factors are boxed
(15, 26). Shown is also the sequence of oligonucleotides used for
bandshift assays and mutagenesis of the IL-8 promoter wild type
(wt), NF-
B mutant (NF-
B mut.),
and NRE mutant (NRE mut.). Point mutations are
underlined. The NF-
B-NREs are shaded.
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Fig. 2.
The NRF-binding site NRE is required for
inducible IL-8 transcription. A, HeLa cells were
transfected with 5 µg of pCSMT3 and 0.25 µg of luciferase reporter
plasmids containing the wild type IL-8 promoter (white bars)
or versions carrying mutations in the NRE (black bars) or
NF- B (hatched bars) elements as described in Fig. 1.
43 h later cells were stimulated with 10 ng/ml IL-1
or left
untreated. 5 h later cells were lysed, and luciferase activity was
determined. Shown are the mean RLU ± S.E. from at least eight
independent experiments. B, HeLa cells were transfected with
2.5 µg of plasmids giving rise to expression of MEKK1, TAK1, TAB1,
MKK62E, and MKK1R4F together with 0.25 µg of
reporter plasmid of wild type (black bars) or mutant NRE
(white bars) IL-8 promoter and 0.5 µg of
pSV-
-galactosidase. Empty pCS3MT (control) was added to a total DNA
amount of 5.75 µg per transfection. 48 h later cells were lysed,
and luciferase activity was determined. Shown is the mean fold increase
compared with the vector control (set as 1) ± S.E. from at least
three independent experiments. C, cells were transfected
with 5 µg of eukaryotic expression plasmids containing no insert
(vector), or cDNAs encoding wild type NRF
(NRFVP16), or the C terminus (amino acids 296-388) of NRF
comprising the DNA binding domain (DBDNRFVP16) fused to the
VP16 transactivation domain together with 0.25 µg of reporter plasmid
and 0.5 µg of pSV-
-galactosidase. 48 h later cells were
lysed, and luciferase activity was determined. Shown is the mean fold
increase compared with the vector control ± S.E. from three
independent experiments.
B-dependent genes through the protein kinases MEKK1
(43) or TAK1 (44). Therefore, we cotransfected the IL-8 promoter
reporter gene together with a constitutively active form of MEKK1 or of
TAK1 together with its coactivator TAB1 (44). This results in
strong induction of the IL-8 promoter (Fig. 2B). Mutation of
the NRE reduced MEKK1- and TAK1-mediated IL-8 transcription by more
than 70% (Fig. 2B). Since MEKK1 and TAK1 activate NF-
B
as well as MAP kinase signaling cascades, we further delineated
the involvement of individual MAPK pathways for
NRE-dependent IL-8 transcription.
B, namely IL-1, MEKK1, and
TAK1. It was thus necessary to characterize the role of protein(s)
binding to the NRE in IL-8 gene expression.
promoter represents the binding site for
NRF (38), we examined if NRF binds to the IL-8 promoter. The minimal
IL-8 promoter-luciferase reporter gene construct was cotransfected with
an effector plasmid encoding an NRF-VP16 fusion protein. This fusion
protein was shown to convert the NRF protein to a transcriptional
activator (38). As shown in Fig. 2C, the IL-8 reporter was
inducible by the simultaneous expression of the NRF-VP16 fusion protein
demonstrating the ability of the fusion protein to bind to the IL-8
promoter. DNA binding of NRF to the IFN-
NRE requires amino acids
296-388 of its C-terminal end (38). This part of the NRF protein is
also sufficient to recognize the IL-8 promoter, as the DBDNRF-VP16
fusion protein stimulated the IL-8 reporter to a similar extent as
NRF-VP16. As a control, coexpression of an empty eukaryotic expression
plasmid had no effect on the transcriptional activity of the IL-8 reporter.
B p65 Binding and
Transactivation--
The IL-8 promoter NRE site overlaps with the
NF-
B site (Fig. 1). NF-
B binding is crucial for inducible
transcription of IL-8. We therefore examined if the impaired induction
of the IL-8 promoter NRE mutant (Fig. 2, A and B)
was caused by a loss of p65 NF-
B binding to the IL-8 promoter.
B as
detected by supershifts (Fig. 3). This indicates that p65 is the major NF-
B subunit binding to the induced IL-8 promoter. This is explained by the fact that the IL-8 promoter NF-
B site lacks the first G of
the NF-
B consensus sequence GGGRNNYYCC, resulting in preferential binding of p65 NF-
B over other REL proteins to this DNA
element as determined by crystallographic studies (48).
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Fig. 3.
Analysis of IL-1-inducible nuclear proteins
binding to the wild type or the NRE mutant IL-8 promoter in
vitro. Nuclear extracts from HeLa cells stimulated for
30 min with 10 ng/ml IL-1 or left untreated were incubated with
radiolabeled wild type (wt) or NRE-mutated (NRE
mut.) IL-8 promoter oligonucleotide. Antibodies against p65
NF-
B (
p65), NRF (
NRF), or preimmune serum (pre Im.) were added to
the binding reactions where indicated. Protein-DNA complexes were
analyzed by EMSA. Two IL-1-inducible complexes are indicated (complex
I and II). III indicates the
constitutive protein-DNA complex, which is not seen when the NRE is
mutated.
B binding to the IL-8 promoter is
not affected by the NRE mutation in vitro (Fig. 3). In cells
transfected with p65 NF-
B, the activity of the wild type IL-8
promoter was induced by 8-fold as compared with cells transfected with
vector alone (Fig. 4). As expected the
p65 NF-
B subunit was unable to induce the mutant NF-
B promoter (Fig. 4). However, the mutant NRE promoter was activated by p65 NF-
B
to the same extent as the wild type promoter (Fig. 4). Taken together
the results presented in Figs. 3 and 4 indicate that the NRE mutation
does not affect DNA binding and transactivation of the IL-8 promoter by
p65 NF-
B.
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Fig. 4.
p65 NF- B
transactivates the IL-8 promoter independent of the NRE. HeLa
cells were cotransfected with 5 µg of empty expression vector
(black bars), 2.5 µg of pCS3MTNRF (hatched
bars), 2.5 µg of pMT7p65 NF-
B (white bars), or 2.5 µg of pCS3MTNRF together with 2.5 µg of pMT7p65 NF-
B
(cross-hatched bars) plus 0.25 µg of luciferase reporter
plasmids containing the IL-8 promoter wild type (wt), or
versions carrying mutations in the NRE (NRE mut.) or NF-
B
elements (NF-
B mut.) and 0.5 µg of
pSV-
-galactosidase. The total DNA amount transfected was kept
constant by adding pCS3MT. 48 h later cells were lysed, and
luciferase activity (RLU) was determined. Shown are the mean RLU ± S.E. from three independent experiments.
B. Instead it correlates with a loss of NRF binding and possibly of additional proteins contained in the constitutive protein DNA complex III (Fig. 3). It is thus possible that in IL-1-stimulated cells
NRF acts as a positive component in the induced promoter.
B and JNK Is Enhanced
by NRF--
Transfection of p65 NF-
B leads to an induction of the
IL-8 promoter (Fig. 4). Cotransfection of NRF did not enhance
transactivation of the IL-8 promoter mediated by transfected p65
NF-
B (Fig. 4). Transfected nuclear p65 NF-
B was far less
efficient in inducing the IL-8 promoter than IL-1 or the protein
kinases MEKK1 and TAK1 (Fig. 2, A and B). This
may be caused by a relatively low amount of transfected NF-
B in the
nucleus, but more likely it indicates the requirement of additional
signals for full IL-8 promoter induction. IL-1, MEKK1, and TAK1
activate other signaling pathways in addition to NF-
B. Any positive
effect of NRF on p65-mediated IL-8 transcription might therefore depend
on the activation of additional pathways that are required for maximal
IL-8 transcription.
B, ERK, JNK, and p38 MAPKs (44,
51-57). MKK6 and MKK1, specific activators of the p38 and ERK MAP
kinases, respectively, activated the IL-8 promoter independently of the
NRE (Fig. 2B). This fact and our previous finding that the
JNK pathway provides a second signal, in addition to NF-
B, for
IL-1-induced IL-8 transcription (41, 42), prompted us to test if the
JNK pathway could provide the putative additional signal.
B. As previously reported by others (59, 60) active MKK7 and JNK2
translocate to the nucleus. We found that the mutant MKK73E
was also present in the nucleus and efficiently activated JNK2 as
assessed by immune complex protein kinase assay (Fig. 5A). Transfected p65 and NRF
did not activate nuclear JNK (Fig. 5A).
View larger version (21K):
[in a new window]
Fig. 5.
NRF cooperates with p65
NF- B and JNK-mediated IL-8 transcription.
A, cells were transfected with pCS3MT (vector),
2.5 µg of pCS3MTMKK73E, 2.5 µg of peVHAJNK2, 2.5 µg
of pMT7p65 NF-
B, 3 µg of pMBC1GFPNRF in the indicated
combinations. DNA amounts were kept constant by adding empty pSC3MT.
24 h later nuclear extracts were prepared. 100 µg of protein
were separated by SDS-PAGE, and epitope-tagged MYC-MKK73E
and HA-JNK2 were detected by Western blotting. JNK was
immunoprecipitated from 250 µg of nuclear extracts with a rabbit
-JNK antibody, and its activity was determined in vitro
using GST-c-JUN (aa 1-135) and [
-32P]ATP as
substrates. Reaction mixtures were separated on SDS-PAGE, and
phosphorylated GST-JUN was visualized by autoradiography. B,
cells transfected as in A were cotransfected with 0.5 µg
of pSV-
-galactosidase and 0.25 µg of luciferase reporter plasmid
containing the wild type IL-8 promoter (black bars) or
versions carrying mutations in the NRE (white bars), NF-
B
(hatched bars), or AP-1 (gray bars) elements. The
total DNA amount transfected was kept constant by adding pCS3MT.
48 h later cells were lysed and luciferase activity (RLU) was
determined. Shown are the mean RLU ± S.E. from four independent
experiments.
B, expression of MKK73E and JNK2 was
sufficient to induce some IL-8 transcription (Fig. 5B, lane
3), which increases when a signal by p65 is provided (Fig.
5B, lane 4). Promoter activation by p65 NF-
B or MKK73E
and JNK2, alone or in combination, was lost when either the NF-
B or
the AP-1 sites were mutated. In agreement with our previous results
(41), this indicates an essential role for both sites for NF-
B and
JNK-dependent IL-8 transcription. Interestingly,
cotransfected NRF enhanced p65 NF-
B and JNK-mediated transcription
(Fig. 5B, lane 6). This positive effect of NRF on IL-8
transcription depends on an intact NRE site, because it was not
observed when the NRE was mutated (Fig. 5B, lane 6). As the
NF-
B and AP-1 promoter mutants failed to respond to NF-
B or
JNK stimuli, the individual contribution of these sites to NRF
coactivation could not be assessed. Together, these data suggest that
simultaneous binding of NF-
B and AP-1 proteins to their respective
promoter elements is a prerequisite for NRF to affect p65 NF-
B and
JNK-mediated IL-8 transactivation.
View larger version (26K):
[in a new window]
Fig. 6.
NRF is required for basal and IL-1-induced
expression of the endogenous IL-8 gene. HeLa tet
transactivator protein cells were stably transfected with
tetracycline-repressible plasmids pTBC containing the coding region of
the NRF cDNA (NRF sense, black bars) or a 300-base pair
fragment of the NRF cDNA in antisense orientation ( NRFas,
cross-hatched bars) or empty pTBC (vector, white
bars) as described under "Experimental Procedures."
Tetracycline (tet) was removed from the culture medium for
48 h to induce NRF sense or antisense mRNA. A,
expression of endogenous NRF protein was analyzed before and after
withdrawal of tetracycline by immunoprecipitation from nuclear cell
extracts followed by Western blotting using
-NRF antibodies.
n.s. indicates nonspecifically detected proteins.
B, the mRNA expression of transfected
neomycin-phosphotransferase (Neo), NRF sense and NRF
antisense (
NRFas) cDNAs, or of endogenous
(end.) IL-8 and NRF genes was analyzed by Northern blotting
of poly(A)+ mRNA before and after withdrawal of
tetracycline. Transcripts were detected by reprobing the membrane with
specific cDNA probes followed by PhosphorImager analysis.
C, cells cultured for 48 h in the presence or absence
of tetracycline were stimulated where indicated with 24 ng/ml IL-1
for 18 h or left untreated. Thereafter IL-8 secreted into the
culture medium was determined by enzyme-linked immunosorbent assay.
Shown is the mean IL-8 concentration ± S.E. measured in four
independent experiments.
promoter (38), constitutive binding of the NRF protein to
the interleukin-8 promoter is responsible for suppression of IL-8 gene
expression in unstimulated cells.
B- and JNK-induced IL-8
transcription in transient transfection assays.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene expression (38).
Sequence analysis revealed that the IL-8 promoter contains an
arrangement of its NF-
B site with a potential binding site for NRF
similar to the corresponding sites in the IFN-
promoter (Fig. 1 and
Refs. 37 and 38). This suggested a homology in regulation of both genes
by NRF. Supershift experiments in EMSA and transfection assays
demonstrate that NRF binds to the IL-8 promoter (Figs. 2 and 3). A
mutant NRE sequence leads to increased transcription from the IL-8
promoter (Fig. 2). Lowering the cellular amount of NRF by antisense
mRNA expression resulted in detectable levels of IL-8 mRNA and
the protein, both of which are absent in unstimulated cells (Fig. 6).
This derepression proves the in vivo participation of NRF in
the constitutive repression of IL-8 and highlights its homologous
function in the regulation of basal promoter activity of the IL-8 and
IFN-
genes.
B and C/EBP as a
consequence of IL-1 stimulation (36). Our study reveals that NRF
represses the IL-8 promoter most likely by a noncompetitive mechanism.
NRF is not replaced by NF-
B after stimulation by IL-1 (Fig. 3), and
as outlined below, it alters its function and is required for maximal
promoter activity during stimulation. We therefore propose that NRF
actively represses transcription factors that bind to the uninduced
IL-8 promoter. Evidence for this mechanism was found for the IFN-
promoter (38).
B is an essential transcription factor for IL-1-induced IL-8 gene expression (15). Overexpressed NRF
did not enhance NF-
B-mediated IL-8 transcription (Fig. 4). This
could indicate that the amount of NRF in the nucleus is saturating. Alternatively, NRF needs further signals from IL-1 induction to function as a coactivator for IL-8 transcription. It was recently found
that the JNK signaling pathway provides an essential signal for
IL-1-induced expression of NF-
B-dependent genes such as
IL-8 and IL-6 (41, 42, 61). When p65 NF-
B was expressed together with active nuclear JNK, cotransfected NRF enhanced IL-8 transcription further (Fig. 5). These results suggest that either NRF is modified itself by a JNK-dependent mechanism or that it is
interacting on the IL-8 promoter with kinase-activated transcription
factor(s). In that context we found that NRF coactivation required not
only an intact NRE site but also binding of NF-
B and AP-1 proteins to their cis-elements (Fig. 5). This suggests that the
binding of all three transcription factors to their DNA-binding sites and their physical interaction is needed for maximal induction of the
IL-8 promoter. The NRE overlaps with the NF-
B site, and indeed, it
was shown previously that NRF is able to interact directly with members
of the REL family (38). NF-
B is modified by protein kinases
(19-21), interacts with various proteins that enhance its transcriptional activity (24, 25, 27, 49), and contacts through its C
terminus components of the basal transcriptional machinery (62). It is
thus possible that in IL-1-stimulated cells NRF enhances NF-
B
activity in concert with AP-1 by acting on one or more of these mechanisms.
is a specific response to viral infection. Most
interesting, NRF in IFN-
regulation only acts as a constitutive repressor (38). After induction by virus, the presence of NRF is not
required for strong IFN-
gene expression, although it remains bound
to the IFN-
promoter (38). Virus induces IL-8 secretion to an extent
similar to IL-1. Preliminary data indicate that virus-induced
expression of endogenous IL-8 was not affected in cell lines expressing
antisense or sense mRNA of
NRF.2 It was recently shown
that cytokines and virus induce IL-8 transcription through distinct
promoter elements (63). Therefore, proteins that interact with NRF at
the IL-8 promoter in an IL-1-dependent manner may be
responsible for the response specificity of the coactivator function of
NRF.
B and
NRE sites is conserved in a variety of genes relevant to inflammation
(37, 65). The NRE does not overlap the NF-
B site in all cases.
However, it was shown previously that the inhibitory effect of NRE to
various NF-
B elements is exhibited over distances up to 2.5 kilobase
pairs (37). Thus, it is tempting to speculate that NRF constitutes a
major transcription factor that prevents uncontrolled expression of
proinflammatory proteins and contributes to their effective synthesis
during disease.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. James R. Woodgett, Jeremy Saklatvala, Natalie Ahn, Jun Ninomiya-Tsuji, Kunihiro Matsumoto, and Eisuke Nishida for the gift of reagents.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants DFG KR1143/2-1, KR1143/2-3, and SFB244/B18 (to M. K.) and the BMBF 0310697 (to H. H.).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: Institute of Pharmacology, Medical School Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. Tel.: 49-511-532-2800; Fax: 49-511-532-4081; E-mail: Kracht.Michael@MH-Hannover.de.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M007532200
2 M. Kracht and M. Nourbakhsh, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
AP-1, activating protein-1;
C/EBP, CAAT enhancer-binding protein;
DTT, dithiothreitol;
EMSA, electrophoretic mobility shift assay;
GST, glutathione S-transferase, HA, hemagglutinin;
JNK, c-Jun-N-terminal kinase;
IFN-, interferon-
;
IP, immunoprecipitation;
MAPK, mitogen-activated protein kinase;
MKK, MAP
kinase kinase;
MEKK1, mitogen-activated protein kinase/ERK kinase
kinase 1;
NF-
B, nuclear factor-
B;
NRF, NF-
B-repressing factor;
NRE, negative regulatory element;
Oct-1, octamer-binding protein-1;
PAGE, polyacrylamide gel electrophoresis;
PMSF, phenylmethylsulfonyl
fluoride;
RLU, relative light units;
TAK1, transforming growth
factor-
-activated kinase 1;
TAB1, TAK1-binding protein 1;
wt, wild
type;
aa, amino acids;
ERK, extracellular signal-regulated
kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baggiolini, M. (1998) Nature 392, 565-568[CrossRef][Medline] [Order article via Infotrieve] |
2. | Baggiolini, M., and Clark-Lewis, I. (1992) FEBS Lett. 307, 97-101[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kasahara, T., Mukaida, N., Yamashita, K., Yagisawa, H., Akahoshi, T., and Matsushima, K. (1991) Immunology 74, 60-67[Medline] [Order article via Infotrieve] |
4. |
Brasier, A. R.,
Jamaluddin, M.,
Casola, A.,
Duan, W.,
Shen, Q.,
and Garofalo, R. P.
(1998)
J. Biol. Chem.
273,
3551-3561 |
5. | Aihara, M., Tsuchimoto, D., Takizawa, H., Azuma, A., Wakebe, H., Ohmoto, Y., Imagawa, K., Kikuchi, M., Mukaida, N., and Matsushima, K. (1997) Infect. Immun. 65, 3218-3224[Abstract] |
6. | Hobbie, S., Chen, L. M., Davis, R. J., and Gala, J. E. (1997) J. Immunol. 159, 5550-5559[Abstract] |
7. | Mastronarde, J. G., Monick, M. M., Mukaida, N., Matsushima, K., and Hunninghake, G. W. (1998) J. Infect. Dis. 177, 1275-1281[Medline] [Order article via Infotrieve] |
8. | Murayama, T., Ohara, Y., Obuchi, M., Khabar, K. S. A., Higashi, H., Mukaida, N., and Matsushima, K. (1997) J. Virol. 71, 5692-5695[Abstract] |
9. |
DeForge, L. E.,
Preston, A. M.,
Takeuchi, E.,
Kenney, J.,
Boxer, L. A.,
and Remick, D. G.
(1993)
J. Biol. Chem.
268,
25568-25576 |
10. | Lee, L.-F., Haskill, S. J., Mukaida, N., Matsushima, K., and Ting, J. P.-Y. (1997) Mol. Cell. Biol. 17, 5097-5105[Abstract] |
11. | Shapiro, L., and Dinarello, C. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12230-12234[Abstract] |
12. |
Sonoda, Y.,
Kasahara, T.,
Yamaguchi, Y.,
Kuno, K.,
Matsushima, K.,
and Mukaida, N.
(1997)
J. Biol. Chem.
272,
15366-15372 |
13. |
Dinarello, C.
(1996)
Blood
87,
2095-2147 |
14. |
Barnes, P. J.,
and Karin, M.
(1997)
N. Engl. J. Med.
336,
1066-1071 |
15. | Mukaida, N., Okamoto, S., Ishikawa, Y., and Matsushima, K. (1994) J. Leukocyte Biol. 56, 554-558[Abstract] |
16. |
Zandi, E.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
4547-4551 |
17. |
Schmitz, M. L.,
dos Santos Silva, M. A.,
and Baeuerle, P. A.
(1995)
J. Biol. Chem.
270,
15576-15584 |
18. | Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597-4607[Abstract] |
19. |
Bird, T. A.,
Schooley, K.,
Dower, S. K.,
Hagen, H.,
and Virca, D.
(1997)
J. Biol. Chem.
272,
32606-32612 |
20. | Zhong, H., SuYang, H., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1997) Cell 89, 413-424[Medline] [Order article via Infotrieve] |
21. | Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661-671[Medline] [Order article via Infotrieve] |
22. |
Van den Berghe, W.,
Plaisance, S.,
Boone, E.,
De Bosscher, K.,
Schmitz, M. L.,
Fiers, W.,
and Haegeman, G.
(1998)
J. Biol. Chem.
273,
3285-3290 |
23. |
Bergmann, M.,
Hart, L.,
Lindsay, M.,
Barnes, P. J.,
and Newton, R.
(1998)
J. Biol. Chem.
273,
6607-6610 |
24. | Stein, B., Baldwin, A. S., Jr., Ballard, D., Greene, W. C., Angel, P., and Herrlich, P. (1993) EMBO J. 12, 3879-3891[Abstract] |
25. |
Gerritsen, M. E.,
Williams, A. J.,
Neish, A. S.,
Moore, S.,
Shi, Y.,
and Collins, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2927-2932 |
26. |
Collins, T.,
Read, M. A.,
Neish, A. S.,
Whitley, M. Z.,
Thanos, D.,
and Maniatis, T.
(1995)
FASEB J.
9,
899-909 |
27. | Merika, M., Williams, A. J., Chen, G., Collins, T., and Thanos, D. (1998) Mol. Cell 1, 277-287[Medline] [Order article via Infotrieve] |
28. |
Oswald, F.,
Liptay, S.,
Adler, G.,
and Schmid, R. D.
(1998)
Mol. Cell. Biol.
18,
2077-2088 |
29. | Plaisance, S., Vanden Berghe, W., Boone, E., Fiers, W., and Haegeman, G. (1997) Mol. Cell. Biol. 17, 3733-3743[Abstract] |
30. | Kannabiran, C., Zeng, X., and Vales, L. D. (1997) Mol. Cell. Biol. 17, 1-9[Abstract] |
31. | Stein, B., and Yang, M. X. (1995) Mol. Cell. Biol. 15, 4971-4979[Abstract] |
32. |
DeBosscher, K.,
Schmitz, M. L.,
VandenBerghe, W.,
Plaisance, S.,
Fiers, W.,
and Haegeman, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13504-13509 |
33. |
Mukaida, N.,
Morita, M.,
Ishikawa, Y.,
Rice, N.,
Okamoto, S.,
Kasahara, T.,
and Matsushima, K.
(1994)
J. Biol. Chem.
269,
13289-13295 |
34. |
Heyninck, K.,
DeValck, D.,
Vanden Berghe, W.,
Van Criekinge, W.,
Contreras, R.,
Fiers, W.,
Haegeman, G.,
and Beyaert, R.
(1999)
J. Cell Biol.
145,
1471-1482 |
35. |
Mukaida, N.,
Shiroo, M.,
and Matsushima, K.
(1989)
J. Immunol.
143,
1366-1371 |
36. |
Wu, G. D.,
Lai, E. J.,
Huang, N.,
and Wen, X.
(1997)
J. Biol. Chem.
272,
2396-2403 |
37. | Nourbakhsh, M., Hoffmann, K., and Hauser, H. (1993) EMBO J. 12, 451-459[Abstract] |
38. |
Nourbakhsh, M.,
and Hauser, H.
(1999)
EMBO J.
18,
6415-6425 |
39. | Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5547-5551[Abstract] |
40. | Finch, A., Holland, P., Cooper, J. A., Saklatvala, J., and Kracht, M. (1997) FEBS Lett. 418, 144-148[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Holtmann, H.,
Winzen, R.,
Holland, P.,
Eickemeier, S.,
Hoffmann, E.,
Wallach, D.,
Malinin, N. L.,
Cooper, J. A.,
Resch, K.,
and Kracht, M.
(1999)
Mol. Cell. Biol.
19,
6742-6753 |
42. |
Krause, A.,
Holtmann, H.,
Eickemeier, S.,
Winzen, R.,
Szamel, M.,
Resch, K.,
Saklatvala, J.,
and Kracht, M.
(1998)
J. Biol. Chem.
273,
23681-23689 |
43. |
Nemoto, S.,
DiDonato, J. A.,
and Lin, A.
(1998)
Mol. Cell. Biol.
18,
7336-7343 |
44. | Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J.-I., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252256 |
45. | Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. (1996) Mol. Cell. Biol. 16, 1247-1255[Abstract] |
46. |
Winzen, R.,
Kracht, M.,
Ritter, B.,
Wilhelm, A.,
Chen, C.-Y. A.,
Shyu, A.-B.,
Müller, M.,
Gaestel, M.,
Resch, K.,
and Holtmann, H.
(1999)
EMBO J.
18,
4969-4980 |
47. | Mansour, S. J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukasawa, K., Vande Woude, G. F., and Ahn, N. G. (1994) Science 265, 966-970[Medline] [Order article via Infotrieve] |
48. | Chen, Y.-Q., Ghosh, S., and Ghosh, G. (1998) Nat. Struct. Biol. 5, 67-73[Medline] [Order article via Infotrieve] |
49. | Stein, B., and Baldwin, A. S. (1993) Mol. Cell. Biol. 13, 7191-7198[Abstract] |
50. | Oliveira, I. C., Mukaida, N., Matsushima, K., and Vilcek, J. (1994) Mol. Cell. Biol. 14, 5300-5308[Abstract] |
51. | Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve] |
52. | Lin, A., Minden, A., Martinetto, H., Claret, F.-X., Lange-Carter, C., Mercurio, F., Johnson, G. L., and Karin, M. (1995) Science 268, 286-290[Medline] [Order article via Infotrieve] |
53. |
Khokhatchev, A.,
Xu, S.,
English, J.,
Wu, P.,
Schaefer, E.,
and Cobb, M.
(1997)
J. Biol. Chem.
272,
11057-11062 |
54. |
Karin, M.,
and Delhase, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9067-9069 |
55. |
Guan, Z.,
Buckman, S. Y.,
Pentland, A. P.,
Templeton, D. J.,
and Morrison, A. R.
(1998)
J. Biol. Chem.
273,
12901-12908 |
56. |
Moriguchi, T.,
Kuroyanagi, N.,
Yamaguchi, K.,
Gotoh, Y.,
Irie, K.,
Kano, T.,
Shirakabe, K.,
Muro, Y.,
Shibuya, H.,
Matsumoto, K.,
Nishida, E.,
and Hagiwara, M.
(1996)
J. Biol. Chem.
271,
13675-13679 |
57. | Yan, M., Dal, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve] |
58. |
Holland, P.,
Suzanne, M.,
Campbell, J. S.,
Noselli, S.,
and Cooper, J. A.
(1997)
J. Biol. Chem.
272,
24994-24998 |
59. |
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674 |
60. | Cavigelli, M., Dolfi, F., Claret, F.-X., and Karin, M. (1995) EMBO J. 14, 5957-5964[Abstract] |
61. | Chu, W.-M., Ostertag, D., Li, Z.-W., Chang, L., Chen, Y., Hu, Y., Williams, B., Perrault, J., and Karin, M. (1999) Immunity 11, 721-731[Medline] [Order article via Infotrieve] |
62. |
Casola, A.,
Garofalo, R. P.,
Jamaluddin, M.,
Vlahopoulos, S.,
and Brasier, A. R.
(2000)
J. Immunol.
164,
5944-5951 |
63. |
Schmitz, M. L.,
Stelzer, G.,
Altmann, H.,
Meisterernst, M.,
and Bauerle, P. A.
(1995)
J. Biol. Chem.
270,
7219-7226 |
64. | Roberts, S. G. E., and Green, M. R. (1995) Nature 375, 105-106[CrossRef][Medline] [Order article via Infotrieve] |
65. | Nourbakhsh, M., and Hauser, H. (1997) Immunobiology 198, 65-72[Medline] [Order article via Infotrieve] |