From the Laboratory of Molecular Biology, Flanders
Interuniversity Institute for Biotechnology and University of Gent,
B-9000 Gent, Belgium and the ¶ Department of Immunochemistry,
German Cancer Research Center, D-69120 Heidelberg, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin-6 (IL-6) is a pleiotropic cytokine,
which is involved in inflammatory and immune responses, acute phase
reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929,
the nuclear factor (NF)-B plays a crucial role in IL-6 gene
expression mediated by tumor necrosis factor (TNF). The levels of the
activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the
human IL-6 gene promoter coupled to a reporter gene, we screened for
possible cooperating transcription factors. Even the smallest deletion
variant, containing almost exclusively a NF-
B-responsive sequence
preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple
B-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a
B-dependent reporter
gene construct, without affecting the levels of NF-
B binding to DNA.
Furthermore, we clearly show that, using a nuclear Gal4
"one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589
have a direct repressive effect on the transactivation potential of the
p65
B subunit. Therefore, we conclude that, in addition to
cytoplasmic activation and DNA binding of NF-
B, the p38 and
extracellular signal-regulated kinase MAPK pathways act as necessary
cooperative mechanisms to regulate TNF-induced IL-6 gene expression by
modulating the transactivation machinery.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interleukin (IL)1 6 contributes to a multitude of physiological and pathophysiological processes. Among its many functions, IL-6 plays an active role in immunological responses, bone metabolism, reproduction, inflammation, neoplasia, and aging. The cellular and molecular biology of IL-6 has been explored by a variety of approaches (1, 2). The regulation of expression of the IL-6 gene is adapted to the key function of this cytokine, namely a systemic alarm signal that recruits diverse host defense mechanisms that serve to limit tissue injury. Inflammation-associated cytokines such as tumor necrosis factor (TNF), IL-1, and platelet-derived growth factor, bacterial products such as endotoxin, and acute viral infections, all enhance IL-6 gene expression. The characterization of the IL-6 promoter revealed a complex control region that can be triggered by multiple activation pathways (3, 4).
In the case of TNF (and of IL-1), the main transcriptional activator
for IL-6 gene induction is the nuclear factor (NF)-B (5-7), which
is typically a dimer between p50 and the transactivating subunit p65
(RelA). In unstimulated cells, NF-
B resides in the cytoplasm. Here,
the DNA-binding dimer is bound to the inhibitory molecule I
B, from
which it is released upon cell stimulation. NF-
B then migrates into
the nucleus, where it effects expression of its numerous target genes
(8).
We have shown previously that in the mouse fibrosarcoma cell line
L929sA, the quantities of activated NF-B do not correlate with the
variations of IL-6 mRNA levels in the cell. We therefore concluded
that other cooperative factors or regulatory mechanisms are necessary
for modulating the levels of NF-
B-dependent IL-6 mRNA production (9).
Studies over the last few years have shown that different
mitogen-activated kinase (MAPK) cascade pathways contribute to the transmission of extracellular signals that can finally result in direct
or indirect phosphorylation of various transcription factors and
alterations in gene expression (10, 11). More particularly, we reported
that abrogation of the p38 MAPK pathway represses TNF-mediated IL-6
gene expression, but not NF-B activation and DNA binding (12).
In the present article, we report on the essential role of NF-B to
trigger IL-6 gene activation in response to TNF in L929sA cells.
Furthermore, we show that, apart from TNF-induced cytoplasmic NF-
B activation and nuclear DNA binding, the TNF-activated p38 and ERK MAPK pathways contribute to transcriptional activation by
modulating the transactivation capacity of the NF-
B p65 subunit.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture, Cytokines, and Inhibitors-- Murine fibrosarcoma L929sA cells (13) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. 24 h before induction, cells were seeded in multiwell dishes such that they were subconfluent at the time of the experiment.
Secreted IL-6 levels were assayed on the basis of the proliferative response of 7TD1 cells (14). Recombinant murine TNF, produced in Escherichia coli and purified to at least 99% homogeneity in our laboratory, had a specific activity of 1.95 × 108 IU/mg of protein, as determined in a standardized TNF cytolysis assay on 164 WEHI cl 13 cells, and contained <24 EU of endotoxin/mg of protein. Reference TNF (code 88/532) was obtained from the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). The pyridinyl imidazole SB203580, a specific inhibitor of the p38 MAPK cascade, was a gift from SmithKline Beecham (15); a 20 mM stock solution was prepared in dimethyl sulfoxide. The inhibitor PD098059, which specifically blocks the ERK pathway, was purchased from Calbiochem-Novabiochem International (San Diego, CA; Ref. 16); a 25 mM stock solution was also prepared in dimethyl sulfoxide. Control experiments showed that the final quantities of organic solvent used did not interfere with any of the assays.Plasmids and Oligonucleotides--
The 1168-base pair human IL-6
promoter was isolated from pGEM1gHIL61 (17) and inserted as a
BamHI-XhoI fragment into an intermediate vector.
A series of 5-terminal deletion variants was generated by subcloning
the respective fragments with the indicated enzymes (Fig. 1) and was
transferred into the multicloning site of pGL3 basic (Promega Biotec,
Madison, WI) in front of the luciferase cDNA, giving rise to the
plasmids p1168hu.IL6P-luc+, p234hu.IL6P-luc+, p110hu.IL6P-luc+ and
p50hu.IL6P-luc+. The recombinant plasmids
p(IL6
B)350hu.IL6P-luc+ and
p(GAL4)250hu.IL6P-luc+ have been described previously
(18). The vector pPGK
GeobpA, constitutively expressing a
neomycin-resistant/
-galactosidase fusion protein under control of
the pPGK promoter from the mouse housekeeping enzyme 3-phosphoglycerate
kinase, was a kind gift of Dr. P. Soriano (Fred Hutchinson Cancer
Research Center, Seattle, WA) (19). The expression plasmids pGal4,
pGal4-p651-551, pGal4-p651-285,
pGal4-p65286-551, pGal4-p65521-551,
pGal4-p65286-521,
pGal4-p65286-551
443-476,
pGal4-p65286-521
443-476, and pGal4-VP16, expressing
the DNA-binding domain of the yeast Gal4 protein either alone, or
fused to various parts of the
B p65 subunit or the transactivation
domain of the viral protein VP16, were described previously (20,
21).
Site-directed Mutagenesis--
Site-directed mutagenesis of the
IL-6 promoter was carried out by the gapped heteroduplex method (22),
using a transformer site-directed mutagenesis kit
(CLONTECH). The following mutator oligonucleotides,
each containing a specific restriction site, were used (altered
nucleotides are underlined): 5-AP1,
5
-ATGCCAAAGTGCTGCAGCACTAATAAAAGAA-3
; CRE,
5
-GCGATGCTAAAGGGATCCACATTGCACAAT-3
; NFIL6,
5
-AAAGGACGTCACAGATATCAATCTTAATAAG-3
; ETS,
5
-CAATCTTAATAAGTCGACCAATCAGCCCCA-3
; GATA,
5
-CTCCAACAAAGATTCTAGAAATGTGG-3
; NF-
B,
5
-CAAATGTGAGATCTTCCCATGAGTCTC-3
; 3
-AP1,
5
-GGGATTTTCCCAGAATTCTCAATATTAG-3
.
Transfection Procedure--
Stable transfection of L929sA cells
was performed by the calcium phosphate precipitation procedure
according to standard protocols (23, 24), using a 10-fold excess of the
plasmid of interest over the selection plasmid pPGKGeobpA.
Transfected cells were selected in 500 µg/ml G418 for 2 weeks, after
which the resistant cell clones were pooled for further experiments. In
this way, the individual clonal variation in expression was averaged,
thus providing a reliable response upon induction. The cotransfected plasmid pPGK
GeobpA, conferring resistance to G418 and expressing constitutive
-galactosidase enzymatic activity, was further used as
an internal control for calculating the protein concentration.
Reporter Gene Analysis--
Luciferase assays were carried out
according to the instructions of the manufacturer (Promega Biotec).
Light emission was measured in a luminescence microplate counter
(TopCount; Packard Instrument Co., Meriden, CT). Luciferase assay
reagent comprised 270 µM coenzyme A and 470 µM luciferin (both from Sigma) plus 530 µM
adenosine triphosphate (Boehringer Mannheim, Mannheim, Germany) in a
reaction buffer containing 10 mM Tricine, 0.54 mM (MgCO3)4Mg(OH)2,
1.34 mM MgSO4, 0.05 mM EDTA, and
16.7 mM dithiothreitol (all from Sigma). Luciferase
activity, expressed in arbitrary light units, was corrected for the
protein concentration in the sample, as indicated, by normalization to
the co-expressed -galactosidase levels or by Bradford's protein
determination (26).
-galactosidase protein levels were quantified
with a chemiluminescent reporter assay Galacto-Light kit (TROPIX,
Bedford, MA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TNF-mediated IL-6 Gene Induction Is Exclusively Dependent on
Activated NF-B--
Several hIL-6 promoter deletion variants and
point mutants were coupled to the luciferase reporter gene (Fig.
1) and stably transfected into the mouse
fibrosarcoma cell line L929sA.
|
|
Involvement of p38 MAPK Pathway in the Transcriptional Activity of
p65--
We have shown previously that inhibition of the p38 MAPK
pathway abrogates TNF-mediated IL-6 gene expression without affecting the levels of TNF-induced NF-B release and DNA binding (12). The
involvement of this pathway in NF-
B-mediated transactivation has now
been further studied using the Gal4 "one-hybrid" technique in
eukaryotic cells, as explained below in more detail. This system has
the advantage that the Gal4-transactivator fusion proteins are
exclusively nuclear and are regulated independently of I
B (20, 27,
28).
|
The p38 and ERK MAPK Pathways Cooperate in the p65 Transactivation
Mechanism in Response to TNF--
In previous studies, the TNF-induced
IL-6-mRNA and protein levels of the endogenous IL-6 gene could be
nearly completely inhibited by SB203580 (12), presumably because of
potentiation of transcriptional effects by post-transcriptional and
translational regulatory events on AUUUA repeated motifs located in the
3-untranslated region of IL-6 mRNA (29, 30). In contrast, at the
transcription level, in absence of this regulatory motifs, using the
luciferase reporter gene system, the activation of p65 by TNF could not
be totally counteracted by SB203580, either on the
NF-
B-dependent promoter constructs (p1168hu.IL-6-Pluc+
and p(IL-6
B)350hu.IL-6-luc+; Fig. 3B) or on
the nuclear fusion protein Gal4-p651-551 (Fig.
3A). Hence, phosphorylation-dependent mechanisms
other than the p38 MAPK cascade activation may also contribute to the TNF-mediated activation of the promoter-transcription complex. In this
respect, we and others have previously shown that the ERK pathway is
also subject to activation by TNF (31, 32). Therefore, we also tested
the effect of the inhibitor PD98059 on TNF-mediated gene activation.
Indeed, PD98059 partially inhibited TNF-mediated gene activation both
for a
B-dependent promoter (Fig.
4B) as well as for the
reporter gene driven by Gal4-p651-551 (Fig.
4A). Moreover, the inhibition by both compounds was additive and decreased TNF-induced activity to basal level without affecting the
levels of the activated NF-
B complex or the expression levels of the
nuclear protein Gal4-p65, respectively (data not shown). Again, these
effects were highly specific for gene activation by NF-
B, in
particular for the transactivator subunit p65, as they did not
affect a different promoter reporter gene system (Fig. 4C).
These data suggest that (at least) two different activated MAPK
pathways cooperate to regulate
B-dependent gene
activation in response to TNF.
|
Role of the Transactivation Domains of p65 in TNF-stimulated
Transcriptional Activity--
It has been reported that the p65
subunit contains at least two strong transactivation domains within its
C terminus. The first domain, TA1, comprises the last 30 amino acids of
p65, whereas TA2 comprises the adjacent 90 amino acids (20). To
delineate the p65 domain, responsible for TNF-mediated transactivation, we tested various fusion proteins of Gal4 coupled to different parts of
the p65 subunit (Fig. 5B).
Fig. 5A shows that, upon TNF treatment, up-regulation of
transactivation was observed with Gal4-p651-551, as was
already shown above, as well as with Gal4-p65286-551
containing an intact C-terminal part of p65. By contrast, activation by
TNF was absent with the variant Gal4-p65286-521, in which
the TA1 domain is missing. This indicates the essential role of the TA1
domain for transactivation by p65 in response to TNF. However,
transactivation could only be partially restored by fusing the TA1
domain to the Gal4 segment (Gal4-p65521-551), indicating
that other protein domains might also be of importance in TNF-mediated
signaling. As a matter of fact, the chimeric form p65286-551443-476, in which the TA1 domain is entirely
present, but which lacks the TA2 domain, also showed a strongly reduced
transactivation capacity, suggesting a cooperative role of the TA2
segment. Indeed, in the construction
Gal4-p65286-521
443-476, where both activation domains
are removed, TNF-induced transactivation was totally abolished.
Evidently, the same was true for Gal4-p651-285, which
lacks the entire C-terminal half of the p65 molecule. These results
defined the two transactivation domains as the protein segments
necessary for mediating the p65 transcriptional activation by TNF.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper, we have focused on the transcriptional activation
by the factor NF-B in response to TNF. By the use of various deleted
and point-mutated versions of the IL-6 promoter, we have documented the
key role of the
B motif for induction by TNF. A number of previous
reports have already described the necessity of cooperation and
association of NF-
B with other DNA-bound transcription factors for
optimal gene activation (33-36). The deletion analysis of the IL-6
promoter shows that such factors have indeed a co-activating and
integrating function for full stimulation of the IL-6 promoter. However, using "loss-of-function" mutants of the IL-6 promoter, the
crucial role of NF-
B is obvious, without the primary need for other
associating DNA-bound factors. Using the "gain-of-function" approach by inserting multiple
B sites in front of an unresponsive promoter, the TNF response could be restored.
Activation of NF-B and its binding to DNA is, however, not
sufficient for IL-6 gene activation by TNF; the requirement of additional activating mechanisms has already been described previously (4, 9). More particularly, recently, we have established the importance
of the p38 MAPK pathway as a necessary mechanism for transcriptional
activity of the IL-6 promoter (12). Our present data with the nuclear
fusion protein Gal4-p65 show that the basal constitutive
transcriptional activity of NF-
B p65, but not that of another acidic
transactivator like VP16, could be specifically enhanced by TNF,
independently of effects involving the cytoplasmic activation of
NF-
B. This increased transcriptional activity is the result of the
activation of MAPK pathways by TNF. The p38 as well as the ERK MAPK
pathway contribute to the specific up-regulation by TNF without
affecting the basal TNF-independent activity. This suggests a
signalization system of at least two steps from the TNF receptor to the
gene, in which, first, NF-
B becomes activated in the cytoplasm by a
I
B
-specific kinase, as recently reported (37), and second, the
"nuclear" transactivation potential of the DNA-bound complex is
modulated by (an) additional phosphorylation event(s) via different
TNF-activated MAPKs. Such a multilevel regulation allows fine tuning
and/or gene specific modulation of the transcriptional activity.
Whether MAPKs act directly on the p65 transcription activation complex,
or via intermediate kinases, remains an open question. Nevertheless, by
in vitro kinase assays it was shown that neither IB
,
nor the NF-
B DNA-binding subunits p50, nor the transactivating C-terminal half of p65 became phosphorylated by p38. However, it is
still conceivable that, in vivo, a kinase downstream in the
p38 and ERK pathways phosphorylates NF-
B subunits (38).
Furthermore, whether MAPK-dependent phosphorylation
effectively takes place on the p65 subunit itself is also not proven. Evidence for phosphorylation-dependent regulation of
NF-B has already been reported by Naumann and Scheidereit (39), who
found increased binding of NF-
B upon phosphorylation of the p65
subunit. Recently, Zhong and co-workers (40) showed a strongly
increased transcriptional activity after phosphorylation of p65 on a
consensus cAMP-dependent protein kinase site, which is
located in the p65 Rel homology domain. However, since this site is
clearly different and distinct from the p65 transactivation domains TA1
and TA2, our data point to another phosphorylation system. Schmitz
et al. (21) also observed increased transcriptional activity
upon treatment of HeLa cells with phorbol ester and suggested a
possible phosphorylation in the p65 TA2 domain by a protein kinase
C-dependent mechanism. It cannot, however, be excluded that
different signals and/or stimuli converge into the same activation
region of the p65 subunit.
Recent data connect transcriptional activity of the B p65 subunit
with the versatile coactivator/cointegrator proteins p300 and CBP (41,
42). Extensive protein-protein interactions have been mapped between
the N- and C-terminal regions of CBP/p300, and the C terminus of p65,
containing both transactivation domains. Interestingly, since our
results with the Gal4-p65 fusion proteins demonstrate a crucial role of
these domains of p65 for TNF inducibility, the possible phosphorylation
status of these domains in p65-CBP interaction may be of particular
interest. Furthermore, the coactivator proteins CBP/p300 are subject
themselves to phosphorylation control and were described as a nuclear
target for S6 kinase pp90rsk and for
cyclin-dependent kinases (43). Other targets for MAPK phosphorylation are part of the RNA polymerase complex (28, 44, 45).
Since RNA polymerase II is constitutively associated with CBP/p300,
interaction of the coactivator with NF-
B in the IL-6 promoter
complex may efficiently recruit the polymerase complex, to trigger
subsequent IL-6 gene expression (46).
In summary, our data show that p38 and ERK MAPK signaling pathways
constitute an additional level of gene regulation by the transcription
factor NF-B, more particularly of the p65 subunit, in response to
TNF. Modulation of the p65 transactivation occurs in the nucleus and
independent from I
B regulation, since it is faithfully reproduced
with the Gal4 fusion proteins. However, whether in vivo the
B p65 subunit itself is a direct or indirect substrate of
TNF-activated p38 and/or ERK MAPK pathways, or else is part of an
integrated transcriptionally active complex, which is subject to
modulation by MAPK phosphorylation, needs further study.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Lee and P. Cohen for
providing SB203580, and Dr. P. Soriano for donating pPGKGeobpA. We
acknowledge F. Cherbal, I. Van Rompaey, and F. Molemans for technical
assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Interuniversitaire Attractiepolen, the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, the Vlaams Actiecomité voor Biotechnologie, and the Vlaams Interuniversitair Instituut voor Biotechnologie.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.
§ Fellow of the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-technologisch Onderzoek in de Industrie.
Research director of the Fonds voor Wetenschappelijk
Onderzoek-Vlaanderen. To whom correspondence should be addressed:
Laboratory of Molecular Biology, K. L. Ledeganckstraat 35, B-9000
Gent, Belgium. Tel.: 32-9-264-51-66; Fax: 32-9-264-53-48; E-mail:
chrish{at}lmb.rug.ac.be.
1 The abbreviations used are: IL, interleukin; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; CBP, cAMP responsive element binding protein binding protein; Tricine, N-tris(hydroxymethyl)methylglycine.
2 W. Vanden Berghe, S. Plaisance, E. Boone, K. De Bosscher, M. L. Schmitz, W. Fiers, and G. Haegeman, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|