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B-Dependent IL-6 Expression in Human Monocytes1
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Division of Hematology, Department of Medicine, and
Division of Developmental Genetics, Department of Biology, University of Groningen, Groningen, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity. Freshly isolated
monocytes treated with the protein phosphatase inhibitor okadaic acid
secreted high levels of IL-6 protein, which coincided with enhanced
binding activity of NF-B as well as with phosphorylation and
activation of the ERK1/2 and JNK proteins. The ERK pathway-specific
inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient
overexpression of inactive mutants of either Raf-1 or JNK1 showed that
both pathways were involved in B-dependent IL-6 promoter activity.
By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did
not affect the DNA binding of NF-B but, rather, acted at the level
of transcriptional activity of NF-B. Interestingly, it was shown
that NF-B-mediated gene transcription, both in the context of the
IL-6 promoter as well as on its own, was dependent on both serine
kinase activity and interaction with c-Jun protein. We conclude that
okadaic acid-induced IL-6 gene expression is at least partly mediated
through the ERK1/2 and JNK pathway-dependent activation of NF-B
transcriptional capacity. Our results suggest that the JNK pathway may
regulate NF-B-mediated gene transcription through its
phosphorylation and activation of c-Jun. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding sites (8). The transcription factor
NF-B has been shown to be a critical regulator of IL-6 gene
transcription (5, 6, 7). Within the 1.2-kb fragment of the 5'-flanking
region of the IL-6 gene the functional NF-B element is located
between positions -73 and -63 (5, 6, 7). NF-B complexes consist of
members of the rel multigene family, which is comprised of
five major proteins: p50, p65 (Rel A), c-Rel, p52, and Rel B (reviewed
in 8 and 9). The most abundant dimer is the p50/p65 NF-B
heterodimer. NF-B is located in the cytoplasm in an inducible form,
in which the heterodimer is complexed to the inhibitory subunit,
IB
. Upon stimulation of the cell, IB is rapidly
phosphorylated and degraded; the released nucleophilic heterodimer then
moves to the nucleus. Both p50 and p65 contribute to NF-B DNA
binding, but only the p65 subunit is responsible for transactivation.
Although simultaneous activity of NF-B and additional transcription
factors such as AP-1 and NF-IL6/CEBP, is required to maximally
induce IL-6 gene transcription, the contribution of the B regulatory
element to the transcriptional activation of the IL-6 gene appears most
prominent (5, 6, 7, 8, 10, 11).
Much progress has been made with respect to the identification of
signal transduction pathways involved in cytokine gene expression. Of
great importance are the so-called mitogen-activated protein kinase
(MAPK) proteins (reviewed in 12 and 13). Activation of the
different MAPK signaling pathways ultimately results in the direct or
indirect phosphorylation and activation of various transcription
factors and alterations in gene expression. Although the involvement of
the MAPK pathways in IL-6 gene regulation in human monocytes is far
from elucidated, there are several observations from other cell systems
that may indicate a functional role for the individual kinase pathways
in activating the transcription factor NF-B. For instance, in COS
cells p90RSK1, the downstream kinase of Raf-1/MEK1/ERK1/2
pathway, has been shown to phosphorylate the N-terminal regulatory
domain of IB both in vitro and in vivo only after
12-O-tetraphorbol 12-myristate 13-acetate
stimulation, and thus enhance NF-B DNA-binding (14). In agreement,
in the lymphoblastoid cell line CEM, overexpression of either MEK1 or
ERK1 demonstrated constitutive nuclear localization of NF-B,
suggesting the involvement of the classical ERK pathway in NF-B DNA
binding activity (15). The c-Jun NH2-terminal
kinase/stress-activated protein kinase (JNK/SAPK) pathway can be
activated by small GTP-binding proteins, including cdc42 and Rac1 (16).
Subsequently, the downstream kinase MEKK1 is activated, which, in turn,
activates MKK4, the upstream kinase of JNK (17, 18). Among the
substrates of JNK are c-Jun, activating transcription factor-2,
and Elk-1. Recently, it was shown that overexpression in T cells of an
active form of MEKK1, a constitutive activator of JNK, results in the
direct activation of the IB kinase, the degradation of IB,
and the activation of an NF-B reporter gene (19). Moreover, in HeLa
cells MEKK1 was shown to directly activate the IB kinase (IKK)
complex in vitro, which, through the phosphorylation of IB, results
in the activation of NF-B (20). These observations indicate a link
between NF-B and the JNK signaling cascade. The question remains,
however, whether the above mentioned signaling pathways are also
operational in monocytes with respect to B-mediated IL-6 gene
transcription.
In the present study we wanted to further identify the involvement of
the ERK1/2 and JNK pathways in mediating B-dependent IL-6 gene
expression in human monocytes. One tool in studies of the importance of
phosphorylation states of signaling molecules is the use of specific
pharmacologic inhibitors of protein phosphatases (PPases). Opposite of
the protein kinases, the PPases regulate the phosphorylation state of
key proteins by removing the phosphate group from the tyrosine, serine,
or threonine residues of activated proteins. Okadaic acid (OA), a
polyether fatty acid, is a potent inhibitor of the phosphoserine and
phosphothreonine phosphatases PP1 and PP2A (21). OA is known to be a
potent stimulator of IL-1 and TNF- production in various cell types,
including monocytic cell lines (22, 23). We investigated the effect of
OA on the ERK1/2 and JNK signaling pathways and their involvement in
NF-B-driven IL-6 expression in human monocytes. Our results provide
evidence for the involvement of the Ras/Raf-1/ERK1/2 pathway, the JNK
pathway, and the c-Jun protein in NF-B-mediated IL-6 gene regulation
in human monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium was purchased from Flow Laboratories (Rockville, MD), FBS was obtained from HyClone (Logan, UT), and Lymphoprep was obtained from Nycomed (Oslo, Norway).
OA was obtained from Sigma (St. Louis, MO). Radionucleotides were
obtained from Amersham (Aylesbury, U.K.). Abs against ERK1, ERK2, and
JNK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and
the anti-CD14 was obtained from Becton Dickinson (Sunnyvale, CA).
The MEK1 inhibitor PD098059 was purchased from New England Biolabs
(Beverly, MA). cDNA probe for IL-6 was provided by Dr. L. Aarden
(Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service, Amsterdam, The Netherlands). GST-c-Jun fusion protein was
provided by Dr. J. Borst (The Netherlands Cancer Institute, Amsterdam,
The Netherlands). ELISA kits for IL-6 (Pelikine Compac kits) were
purchased from CLB (Amsterdam, The Netherlands), and anti-TNF-
was obtained from Boehringer Mannheim (Mannheim, Germany). The
luciferase detection kit was purchased from Promega (Madison, WI), and
IL-3 was received from Genetics Institute (Cambridge, MA). All IL-6
promoter constructs, containing various portions of the human IL-6
promoter cloned into the pGL2 luciferase reporter vector
(Promega), were generated in our laboratory. pGAL4-p65 was provided by
Dr. P. A. Baeuerle (Tularik, San Francisco, CA); pGAL4-dbd and
pGAL4tkluc were provided by Dr. S. Wissink (NIOB, Utrecht, The
Netherlands). pRSV-N
Raf1 was provided by Dr. P. J. Coffer
(Department of Pulmonary Diseases, University Hospital Utrecht,
Utrecht, The Netherlands), pcDNA3-Flag-JNK was provided by Dr. R.
J. Davis (Howard Hughes Medical Institute, University of Massachusetts
Medical School, Worchester, MA). pCMV-TAM67 was provided by Dr. M.
J. Birrer (Biomarkers and Prevention Research Branch, National Cancer
Institute, National Institutes of Health, Rockville, MD). The plasmid
p(NF-B)3xCAT was provided by Dr. J. Hiscott (The Sir Mortimer B.
Davis-Jewish General Hospital, Montreal, Canada).
Preparation of monocytes and cell culture
Peripheral blood cells were obtained from healthy volunteer platelet donors, and mononuclear cell suspensions were prepared by Ficoll-Hypaque density-gradient centrifugation. T lymphocytes were depleted by 2-aminoethylisothiouronium bromide-treated SRBC rosetting. Monocytes were further enriched by plastic adherence (1 h, 37°C) and demonstrated a purity of >95%, detected by FACS analysis with anti-CD14 Ab. Monocytes were cultured at 37°C at a density of 1–2 x 106/ml in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 6 ng/ml colistine, and the appropriate amount of FBS. The cell line employed in these studies was TF-1, a human erythroleukemia cell line (24). The TF-1 cell line was cultured in RPMI 1640 supplemented with 5% FBS and 10 ng/ml IL-3. The toxicity of OA was assessed by trypan blue exclusion assays.
IL-6 protein determination
Freshly isolated monocytes (1 x 106 cells in 1
ml) were incubated in RPMI 1640 with 2% FBS. Sixteen hours after the
adherence step, medium was replaced by 1 ml of fresh RPMI 1640
containing 2% FBS, and cells were subsequently stimulated. Twenty-four
hours after treatment with medium or OA (30 ng/ml) the cell-free
supernatants were collected and analyzed for secreted IL-6 protein by
ELISA. Experiments with the MEK1 inhibitor PD98059 were performed by
pretreatment of monocytes for 30 min with 10 µM PD98059 before
stimulation with medium or OA (25). Similarly, monocytes were
preincubated with TNF- or IL-1ß Ab before OA stimulation (1/1000
diluted polyclonal antiserum against TNF- or IL-1ß was maximally
effective, as studied with in vitro dose-response curves).
Nuclear run-on assay
The run-on analysis was performed as previously described using 40 x 106 monocytes/reaction (26). Freshly isolated monocytes were cultured in RPMI 1640 medium supplemented with 2% FBS for 16 h before stimulation with medium or medium plus OA for 1 or 7 h (30 ng/ml).
Five micrograms of the following DNAs were immobilized on Hybond N+ membranes (Amersham): 1) EcoRI-linearized pGEM (negative control), 2) EcoRI-linearized plasmid containing a 1.3-kb fragment of the rat GAPDH cDNA, and 3) EcoRI-linearized plasmid containing a 0.3-kb fragment of the human IL-6 cDNA. Hybridization of labeled RNAs to these membranes was performed at 65°C for 18 h in 0.5 mol/l Na2HPO4 (pH 7.2), 1 mM EDTA, and 7% SDS. Membranes were then washed as described previously (26) and exposed to Kodak X-OMAT XAR films (Eastman Kodak, Rochester, NY) at -80°C using an intensifying screen. Quantitation of the newly synthesized RNAs was performed by phosphorimaging (PhosphorImager, Molecular Dynamics, Sunnyvale, CA), after normalization to the GAPDH signal.
Electrophoretic mobility shift assay (EMSA)
After overnight culture in RPMI 1640 supplemented with 2% FBS
monocytes were stimulated, and nuclear extracts were prepared according
to the miniscale procedure previously described (27). Nuclear extracts
were divided into small aliquots and were stored at -80°C.
Double-stranded synthetic oligonucleotide probes containing the NF-B
consensus sequences (NF-B, 5'-AGCTGCGGGGATTTTCCCTG-3')
was used in the gel retardation assay. The consensus sequence for
binding of the nuclear factor is underlined. Fifty nanograms of
HPLC-purified single-stranded oligonucleotide was labeled with
T4-polynucleotide kinase and [
-32P]ATP (3000 Ci/mmol;
Amersham), separated from nonincorporated radiolabel by Sephadex G-50
chromatography, ethanol precipitated, dried, and dissolved in 20 µl
of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 1 mM
EDTA, and 1 mM DTT, containing a 4-fold excess of the opposite strand.
Annealing of the two strands was performed by heating the mixture for 2
min at 90°C and slow cooling to room temperature. Five micrograms of
nuclear extract and 0.1 ng of double-stranded labeled oligonucleotide
were incubated in 20 mM HEPES (pH 7.9), 60 mM KCl, 0.06 mM EDTA, 0.6 mM
DTT, 2 mM spermidine, and 10% glycerol supplemented with 2 µg
poly(dI-dC). The binding reaction was performed at 26°C for 25 min.
Competition experiments were performed by adding a 100-fold molar
excess of unlabeled self or nonself double-stranded oligonucleotides.
Supershift experiments were performed by incubating the nuclear
extracts and labeled oligonucleotide with polyclonal Abs (1 µg)
against the p50, p65, c-Rel, and p52 subunits of NF-B (Santa Cruz
Biotechnology). The samples were loaded on prerun (30 min, 100 V) 4%
(30/1) polyacrylamide gels and run for 1 h at 150 V in 0.5x TBE
at room temperature. Gels were dried and exposed to Kodak XAR films at
-80°C with an intensifying screen. Quantification of protein binding
was performed by densitometry using a PhosphorImager (Molecular
Dynamics).
Transient transfection assays
The luciferase reporter plasmids pIL6luc(-602), pIL6luc(-122),
and pIL6luc(-60), containing various portions of the human IL-6
promoter cloned into the pGL2 luciferase reporter vector
(Promega), were transfected into TF-1 cell line by means of
electroporation. Before transfection, cells were cultured for 16 h
at a density of 0.5 x 106 cells/ml in the appropriate
medium, washed twice, and resuspended in RPMI 1640 at a density of
10 x 106 in 200 µl. When transfected with a single
plasmid 25 µg of DNA was added, and the mixture was left at room
temperature for 15 min. Cotransfections were performed with 15 µg of
the reporter plasmid (pIL6luc(-602), pIL6luc(-122), or
p(NF-B)3xCAT) together with 15 µg of the expression plasmid
(pRSV-NRaf1, pcDNA3-Flag-JNK1, pCMV-TAM67, or pcDNA3 empty vector).
Cotransfections of pGAL4tkluc (5 µg) with either pGAL4-p65 (5 µg)
or pGAL4-dbd (5 µg) were performed under similar conditions.
Electroporation, in 0.4-cm electroporation cuvettes, was performed at
240 V and 960 µF with a Gene Pulser electroporator (Bio-Rad,
Richmond, VA). After electroporation the cells were replated in RPMI
1640 containing 2% FBS. Six hours after transfection cells were
stimulated for 24 h with medium or OA (30 ng/ml). The cells
were then harvested and lysed by commercially available luciferase
lysis buffer. One hundred microliters of lysis product were added to
100 µl of luciferase assay reagents, and luciferase activity was
measured with the Anthos Lucy1 luminometer (Anthos Labtec Instruments,
Salzburg, Austria).
Western blotting for ERK1 and ERK2
Phosphorylation of ERK1 and ERK2 was analyzed by Western blotting. Briefly, monocytes were cultured for 16 h in RPMI 1640 containing 0.1% FBS and subsequently stimulated for various periods of time with medium or OA (30 ng/ml). After harvesting, total cell extracts were prepared by resuspending the cells in 500 µl of 1x sample buffer (containing 2% SDS, 10% glycerol, 2% 2-ME, 60 mM Tris-Cl (pH 6.8), and bromphenol blue) and lysing the cells by passing them through a 23G1 needle (three times). Cell extracts were directly boiled for 5 min and were stored at -20°C. Before loading, samples were again boiled for 5 min, and cell extracts were resolved by running 0.1 vol on a SDS-12.5% PAGE gel (acrylamide/bisacrylamide, 173/1) and transferred to cellulose nitrate membrane (Schleicher & Schuell, Germany). Immunoblotting with anti-ERK2 Ab was performed by standard procedures, and detection was performed according to the manufacturer’s guidelines (ECL, Amersham). After ERK2 band-shift detection the membrane was stripped (strip buffer containing 1x PBS, 0.1% Tween, and 0.1% SDS) and rehybridized with anti-ERK1 Ab.
Bacterial expression of GST fusion proteins
Bacterial expression of GST-c-Jun has been described previously
(28). Briefly, GST fusion proteins were expressed in Escherichia
coli DH5, induced with 1 mM
isopropyl-ß-D-thiogalactopyranoside and purified with
glutathione-Sepharose beads.
Immunoprecipitation and kinase activity assays for JNK
JNK kinase activity was determined by the ability of this enzyme
to phosphorylate its substrate proteins c-Jun in the presence of
[-32P]ATP. Before stimulation monocytes were cultured
for 16 h in RPMI 1640 plus 0.1% FBS. Cells (15 x
106) were then treated for various periods of time with
medium or OA (30 ng/ml). After stimulation, cells were harvested and
resuspended in 400 µl of lysis buffer (20 mM HEPES (pH 7.4), 2 mM
EGTA, 50 mM ß-glycerophosphate, 1 mM DTT, 1 mM
Na3VO4, 1% Triton X-100, 10% glycerol, 10
µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 0.4
mM PMSF) and lysed on ice for 15 min. After centrifugation at 3000 rpm
and normalization for protein content in the supernatant, lysates were
incubated with anti-JNK Ab in a total volume of 500 µl and
rotated for 30 min at 4°C. Twenty-five microliters of a slurry of
50% protein A-Sepharose beads was then added to the lysate/Ab mixture
and left rotating overnight at 4°C. Subsequently, the
Ab/beads/conjugate mixture was washed with lysis buffer (three times),
twice with LiCl buffer (500 mM LiCl, 100 mM Tris-Cl (pH 7.6), 0.1%
Triton X-100, and 1 mM DTT), and finally with buffer A (three times; 20
mM MOPS (pH 7.2), 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, and
0.1% Triton X-100). After supernatant was completely removed, the
reaction was initiated by adding 50 µl of reaction mix
(43.5 µl of buffer A, 20 mM MgCl2, 25 µM ATP, 3.5 mg/ml
GST-c-Jun, and 10 µCi of [-32P]ATP), and incubation
took place at 20°C for 30 min. The reaction was terminated by adding
15 µl of sample buffer/2-ME (four times). Before resolving samples on
SDS-PAGE gel, samples were boiled for 5 min. A m.w. marker was used to
assess the correct protein size. After running, the gel was
washed in water for 15 min, in 5% TCA/1% sodium pyrophosphate (twice,
2 h each time), and again in water, and subsequently the gel was
dried. Phosphorylated substrate was visualized by autoradiography and
was quantified by densitometric scanning.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly isolated human monocytes were stimulated with the
serine/threonine-specific PPase inhibitor OA and after 24 h of
stimulation IL-6 protein levels were measured in cell-free
supernatants. Treatment of monocytes with OA (30 ng/ml) resulted in
distinct increases in the secretion of IL-6 protein (1898 ± 1164
vs 4 ± 4 pg/ml for unstimulated control; p <
0.005; Fig. 1
). Treatment of the cells
with higher concentrations of OA, up to 200 ng/ml, induced even higher
levels of IL-6 protein. These concentrations, however, appeared toxic
after 24 h of stimulation. Additional experiments were thus
performed with 30 ng/ml OA.
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from human
monocytes (23). To examine whether the effects of OA on IL-6 secretion
are mediated directly or indirectly through the release of TNF-, Ab
against this cytokine was added to the cell culture. Fig. 1
illustrates
that treatment of monocytes with OA plus anti-TNF- resulted in a
partial down-regulation of IL-6 secretion compared with that after
treatment with OA alone (1104 ± 628 pg/ml for OA plus
anti-TNF- vs 1898 ± 1164 pg/ml for OA alone;
p < 0.005), indicating that the secretion of IL-6
protein by OA is only partly an indirect phenomenon mediated by the
release of TNF-. Addition of an anti-IL-1ß Ab had no effect
(data not shown).
To determine whether the OA-mediated increase in IL-6 protein secretion
was established at the transcriptional level, nuclear run-on studies
were performed. Monocytes were treated with medium or medium plus OA
(30 ng/ml) for 7 h, after which the IL-6 in vitro transcription
rate was determined. Results demonstrated that after normalization for
the GAPDH signal, OA enhanced the IL-6 transcription rate 3.2-fold
after 7 h of stimulation (Fig. 2).
The increase in the in vitro IL-6 transcription rate was quantified by
phosphorimaging. The up-regulated IL-6 transcription rate was reflected
at the level of mRNA. Treatment of monocytes with OA (30 ng/ml) for
various periods of time (0–10 h) resulted in enhanced IL-6 mRNA
levels, as determined by Northern blot analysis (data not shown). IL-6
mRNA was first observed after 6 h of stimulation with OA. The slow
activation kinetics of OA, as observed in the nuclear run-on and
Northern analysis, are in agreement with previous reports describing
slow activation of both TNF- and IL-1ß gene expression after OA
stimulation (29, 30).
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B
To delineate the role of the transcription factor NF-B in
OA-mediated IL-6 expression we performed EMSAs using nuclear extracts
from OA-stimulated monocytes. In unstimulated monocytes binding of the
NF-B complex was low (Fig. 3,
lane 1), whereas stimulation with OA (30 ng/ml) for 4 or
6 h resulted in a distinct increase in NF-B binding activity
(Fig. 3, lanes 2 and 4, respectively). Exposure
to OA for shorter periods did not enhance NF-B binding activity
(data not shown). Supershift experiments performed with Abs specific
for the p50 and p65 subunits of the NF-B family indicated that in
monocytes the NF-B complex, which binds to the B-specific binding
sequence, indeed consisted of the p50 and p65 subunits, both before and
after OA exposure (Fig. 4A,
lanes 1–3 and 4–6, respectively). It is well
established that NF-B consisting of p50 and p65 can be
transcriptionally active (8, 9). Two other members of the NF-B
family, c-Rel and p52, were not identified in the OA-induced NF-B
complex (Fig. 5).
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The MAPK signaling cascade involving Raf-1, MEK1, and ERK1/2
phosphorylation is probably an important signal transduction pathway
leading to cytokine gene expression (12, 13). To study the effects of
OA on activity of this MAPK pathway, we measured the phosphorylation of
ERK1 (p44MAPK) and ERK2 (p42MAPK). Monocytes
were treated with OA (30 ng/ml) for various periods of time and were
subsequently analyzed for phosphorylated ERK1 and ERK2 by means of
immunodetection. As shown in Fig. 6, OA
induced a shift in the mobility of both ERK1 and ERK2, indicating
phosphorylation of the respective proteins. The observed shift,
however, demonstrated slow kinetics. The ERK1 shift was first seen
after 30 min of stimulation with OA (data not shown), and
phosphorylation peaked at 90 min of stimulation. The ERK2 shift was
first observed after 60 min of stimulation, and an optimal
phosphorylation state was found at 90 min. In contrast, PMA-mediated
ERK phosphorylation peaked as early as 5 min after addition of the
stimulus. In view of the slow activation kinetics of the ERKs after OA
treatment, we treated monocytes with OA and anti-TNF- Ab
simultaneously and subsequently analyzed for ERK phosphorylation.
Anti-TNF- coincubation did not affect the OA-induced ERK shifts
(data not shown). In this respect, delayed onset of OA-induced activity
was described previously for OA-mediated ERK activity in Jurkat T cells
and B lymphocytes (31, 32). A relevant explanation for the delayed
onset is that OA elicits its effect by disrupting the balance between
low, basal protein phosphorylation and basal protein dephosphorylation
in favor of protein phosphorylation. Signaling modules will be
subsequently activated only after a relatively long incubation time.
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, OA enhanced JNK1
activity as early as 15 min after stimulation. JNK1 kinase activity
peaked at 90 min of stimulation with OA.
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B DNA
binding activity
To summarize, we demonstrated that OA up-regulated IL-6 protein
and mRNA levels in monocytes, which coincided with enhanced binding
activity of NF-B. Furthermore, the MAP kinases ERK1, ERK2, and JNK1
were activated in response to OA treatment. The question remains,
however, whether the OA-induced phosphorylation of ERK1, ERK2, and JNK1
is directly involved in the up-regulation of IL-6 gene expression by
OA. To answer this question with regard to the ERK1/2 pathway we
treated monocytes with the Raf-1/MEK1/ERK pathway-specific
pharmacological inhibitor PD98059. At a concentration of 10 µM
PD98059 the activity of MEK1, the kinase upstream from ERK1 and ERK2,
was almost totally inhibited (25). In our experiments PD98059
dramatically down-regulates OA-induced IL-6 secretion (100% for
OA-induced IL-6 secretion vs 7.3 ± 4.1% for PD98059- plus
OA-mediated IL-6 secretion; p < 0.005; Fig. 8), indicating that OA-enhanced
phosphorylation of ERK1 and ERK2 is an important step in the elevation
of IL-6 protein levels. PD98059 at this concentration was not toxic for
the monocytes, as determined by trypan blue exclusion assays and
lactate dehydrogenase levels in culture supernatants (data not shown)
(33).
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B
(or of AP-1 or NF-IL6; data not shown), as determined after
phosphorimaging quantification (Fig. 3, lanes 3 and
5). Thus, the ERK pathway did not play a role in DNA binding
activity of this transcription factor. We, subsequently, demonstrated
that the observed difference between inhibited IL-6 protein secretion
and the lack of effect regarding NF-B binding activity by PD98059
could not be explained by a change in composition of the NF-B
complex. The NF-B complex consisted of p50 and p65 subunits both
with and without PD98059 pretreatment (Fig. 4A, lanes
4–9). OA enhances IL-6 promoter activity in the TF-1 myeloid cell line
IL-6 transcription was investigated in more detail by transiently
transfecting the TF-1 cell line with three 5'-end deletion constructs
derived from the human IL-6 promoter linked to the luciferase reporter
gene, pIL6luc(-602), pIL6luc(-122), and pIL6luc(-60) (Fig. 9A). Results from the
transfection experiments demonstrated that, in concordance with the
observations in monocytes, OA induced activity of the entire IL-6
promoter in TF-1 cells (Fig. 9B). After transfection with
the largest promoter construct pIL6luc(-602), which contains the
binding sites for both AP-1 and NF-B, OA (30 ng/ml) up-regulated
IL-6 promoter activity 7.8 ± 4.8-fold (p
< 0.05). When pIL6luc(-112), which merely contains the NF-B
binding site, was introduced into TF-1 cells, OA elicited a 14.3
± 10.0-fold (p < 0.05) induction in IL-6
promoter activity. This would imply possible negative regulatory
cis elements in the promoter region downstream from the
NF-B site, within the region -122 through -602. Using a promoter
construct lacking the binding sites for any of the transcription
factors (pIL6luc(-60)), a minimal residual induction of the promoter
activity was observed after OA treatment (1.5 ± 0.3-fold). These
results indicated that OA is a potent inducer of IL-6 gene
transcription, and furthermore, that transactivation by the NF-B
complex greatly contributes to OA-induced IL-6 gene transcription.
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B, TF-1 cells were transfected with the pGAL4-p65 or
pGAL4-dbd constructs in combination with the reporter plasmid
pGAL4tkluc (34). The GAL4-transactivator fusion proteins are
exclusively nuclear and are regulated independently of IB. The
reporter plasmid pGAL4tkluc is under the control of multiple GAL4
binding sites. This GAL4 one-hybrid technique allows analysis of the
B p65 transactivation mechanism. The results demonstrated that OA
enhanced p65-mediated transactivation by 2.2 ± 1.2-fold
(p < 0.05), indicating a direct effect of OA
on transcriptional activity of the p65 subunit of the NF-B complex
(Fig. 9C). Transfection with the negative control plasmid
pGAL4-dbd showed no enhanced promoter activity after OA treatment.
Inactive mutants of Raf-1 and JNK1 down-regulate NFB-mediated
promoter activity
Since previous reports on the transcriptional activation of the
IL-6 gene have emphasized the indispensability of the NF-B binding
site for the activity of the IL-6 promoter, we focussed on the
involvement of the ERK1/2 and JNK1 pathways in NF-B-dependent IL-6
promoter activity (5, 6, 7). For this purpose TF-1 cells were transiently
transfected with pIL6luc(-122), comprising the NF-B binding site
only, together with either pRSV-NRaf1 or pcDNA3-Flag-JNK1, before
stimulation with OA. Previously, pRSV-NRaf1, encoding a dominant
negative mutant of Raf-1, was shown to block mitogen-activated protein
kinase activity by growth factors (35). pcDNA3-Flag-JNK1 encodes a
catalytically inactive mutant of JNK1 (36). The results of these
experiments are depicted in Fig. 10A. Both pRSV-NRaf1
(10 ± 8% for pRSV-NRaf1 vs 100% for pcDNA3;
p < 0.05) and pcDNA3-Flag-JNK1 (54 ± 32% for
pcDNA3-Flag-JNK1 vs 100% for pcDNA3; p < 0.05)
inhibited NF-B-mediated IL-6 promoter activity. Moreover,
cotransfections of the pCMVluc plasmid, containing a constitutive
promoter, with either pRSV-Nraf1 or pcDNA3-Flag-JNK1 did not affect
constitutive promoter activity. These observations emphasize the
specificity of the dominant negative MAP kinase constructs in
inhibiting IL-6 promoter activity.
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B itself and not with possible
regulatory elements in the vicinity of the NF-B binding site, TF-1
cells were simultaneously transfected with pRSV-NRaf1 or
pcDNA3-Flag-JNK1 and p(NF-B)3xCAT. This CAT reporter plasmid solely
contains three consecutive GGGAAAATCC B binding sites adjacent to
the SV40 promoter. Again, it was demonstrated that both pRSV-NRaf1
and pcDNA3-Flag-JNK1 impaired OA-induced B-mediated gene
transcription (58 ± 11% for pRSV-NRaf1 vs 100% for pcDNA3
(p < 0.05) and 54 ± 24% for
pcDNA3-Flag-JNK1 vs 100% for pcDNA3 (p <
0.05); Fig. 10B). Taken together, these data would implicate
a role for both the Raf-1/MEK1/ERK and the MEKK1/JNK pathways in
enhancing the NF-B transactivation capacity.
NF-B-mediated transactivation of the IL-6 promoter, but not
NF-B DNA binding activity, is serine kinase dependent
With the described results we demonstrated that both the MEKK1/JNK
and Raf-1/MEK1/ERK pathways are involved in enhancing NF-B-mediated
promoter activity, but the latter pathway does not affect OA-induced
NF-B DNA binding. This observed difference would imply an activation
signal different from mere binding of NF-B to its consensus
sequence. To resolve whether serine kinase activity may be involved in
this process, transiently transfected TF-1 cells with pIL6luc(-122)
were treated with the serine kinase-specific inhibitor H7 30 min before
OA stimulation. Fig. 11 is a
representative of two independent experiments. H7 was capable of
reducing basal pIL6luc(-122) promoter activity. More importantly,
however, OA-induced pIL6luc(-122) promoter activity was blocked 10- to
100-fold by H7, which coincided with dramatically reduced IL-6 protein
secretion levels in monocytes (Fig. 8). In contrast, band-shift assays
demonstrated that the binding of NF-B to its consensus sequence was
not altered due to H7 pretreatment, implying an essential role for
serine kinase activity in regulating the transactivation potential of
NF-B (Fig. 3, lane 6). H7 pretreatment did inhibit AP-1
binding activity, indicating differential signaling modules involved in
binding of NF-B and AP-1 (data not shown).
|
B-mediated IL-6
promoter activity
Recent work has indicated that Rel family members may interact
with other transcription factors to elicit transactivation of genes.
One such example is the AP-1 complex (8, 37). Since blocking the JNK
pathway inhibited transactivation potential of NF-B, we investigated
whether interaction of NF-B with c-Jun, an important downstream
target of JNK1, plays a role in transcriptional activation of the
NF-B complex. For this purpose, we transfected TF-1 cells with the
pIL6luc(-122) construct together with pCMV-TAM67, the N-terminally
truncated dominant negative c-Jun expression vector (38). The
effectivity of pCMV-TAM67 was verified using a reporter containing five
consecutive AP-1 binding sites (p(TRE)5xCAT). The AP-1 transcription
factor is composed of c-Jun and c-Fos subunits (39). Our experiments
confirmed that reporter activity was indeed inhibited when p(TRE)5xCAT
was cotransfected with pCMV-TAM67 (data not shown). OA-induced
NF-B-driven IL-6 promoter activity was dramatically down-modulated
after introduction of pCMV-TAM67 (100% and 5.9 ± 0.5%;
p < 0.05, respectively), implying a role for c-Jun in
the transcriptional potential of NF-B (Fig. 10A). To
confirm that the effect elicited by pCMV-TAM67 indeed involved the
NF-B complex and not possible regulatory proteins in the vicinity of
the NF-B site, TF-1 cells were transfected with pCMV-TAM67 and
p(NF-B)3xCAT simultaneously. Overexpression of dominant negative
c-Jun again strongly inhibited NF-B-mediated promoter activity
(28 ± 7% for pCMV-TAM67 vs 100% for pcDNA3; p
< 0.05; Fig. 10B). These results strongly suggest that
c-Jun cooperates with NF-B in activating a NF-B-driven promoter
element.
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Both ERK1/2 and JNK
belong to the family of MAPKs that are important mediators of signal
transduction from cell surface receptors to the nucleus. The three
classical parallel MAP kinase cascades have been identified to date are
mediated through ERK1/ERK2 (p44MAPK and
p42MAPK), JNK (SAPK1), and p38 (RK, CSBP, MPK2) (12, 13).
In our studies, pretreatment of monocytes with PD98059, a specific
inhibitor of MEK1, the upstream kinase from ERK1/2, showed an
inhibition of OA-induced IL-6 protein secretion from monocytes, which
was not reflected by a change in NF-B DNA binding activity (25).
Although this would implicate involvement of the MEK1/ERK1/2 pathway in
IL-6 protein secretion, this inhibition was not accomplished by
altering the DNA binding properties of NF-B. This observation in
monocytes is contradictory to reports in other cell systems describing
a close association between ERK1 activity and phosphorylation and
degradation of IB protein, thus leading to NF-B activation.
Sonoda et al., for instance, reported in vitro phosphorylation of
GST-IB- by OA-activated ERK1 (41). Studies by Schouten et al.
suggested that the mitogen-activated 90-kDa ribosomal S6 kinase
(p90rsk1) is an IB kinase (42). p90rsk1,
which is a serine/threonine kinase downstream from the Raf/MEK1/ERK1/2
cascade, was shown to phosphorylate IB on two serine residues,
i.e., Ser32 and Ser36. Moreover, in the
lymphoblastoid cell line CEM, overexpression of either MEK1 or ERK1
demonstrated constitutive nuclear localization of NF-B, suggesting
involvement of the classical ERK pathway in NF-B DNA binding
activity (15). In our cell system, however, it is unlikely that OA
activates ERK1, which, in turn, phosphorylates IB, followed by the
degradation of IB. Rather, IB is phosphorylated due to OA
stimulation of a pathway distinct from the Raf-1/MEK1/ERK pathway.
Recently, two upstream kinases of IB have been cloned, IKK and
IKKß (IB kinase) (43, 44). It was shown
that OA inhibited a PP2A, which normally dephosphorylates IKK, thus
leading to enhanced IB phosphorylation and degradation and
enhanced NF-B binding activity (45). This signaling route may,
independently from the Raf-1/MEK1/ERK1/2 pathway, account for the
enhanced NF-B DNA binding activity as observed after OA stimulation.
Although the Raf-1/MEK1/ERK1/2 pathway did not affect NF-B DNA
binding activity, we clearly demonstrated that overexpression of an
inactive mutant of Raf-1 inhibited NF-B-mediated IL-6 promoter
activity. This indicates that the ERK pathway serves as an additional
signaling pathway that is distinct from IB degradation, NF-B
nuclear translocation, and DNA binding, but is required for NF-B
transcriptional activation by OA. In agreement with this observation
are previous reports describing additional regulatory mechanisms, other
than modulating NF-B DNA binding properties, that enhance the
transcriptional activity of NF-B. In this respect, serine
phosphorylation of the p65 subunit of NF-B as a regulatory step in
the transcriptional activity of NF-B has been reported. Indeed, in
our setting OA was capable of enhancing p65-mediated transcription from
the GAL4 promoter. The p65 subunit contains at least two strong
transactivation domains within its C terminus (46). The first domain,
TA1, is contained within the last 30 amino acids of p65, whereas TA2
comprises the adjacent 90 amino acids. Schmitz et al. reported that
phosphorylation and transcriptional activity of a defined region within
the TA2 domain were stimulated by phorbol ester treatment of cells, and
thus augmented the transactivation potential of NF-B (46). In a
recent study the catalytic subunit of protein kinase A was shown to
phosphorylate p65 at a serine residue 276, thus increasing the
transactivational capacity of NF-B without affecting nuclear
translocation (47). Subsequently, it was demonstrated that as a result
of protein kinase A-mediated phosphorylation, p65 creates a site for
interaction with the transcriptional coactivator CBP/p300, which
results in enhanced transcriptional activity (48, 49). In fibroblasts
and hepatoma cells IL-1-mediated NF-B activation was accompanied by
casein kinase II-mediated phosphorylation of the p65 subunit on serine
residues (50). These data are in accordance with the results of our
experiments showing that NF-B transactivation capacity, but not DNA
binding, is dependent on serine kinase activity. In our experimental
setting, however, it remains unclear whether the MAPK-dependent
phosphorylation effectively takes place on the p65 subunit itself. In
this regard, we demonstrated that the JNK pathway also mediated the
transcriptional potential of NF-B, both in context of the IL-6
promoter and when bound to individual NF-B binding sites. The JNK
protein kinases phosphorylate the NH2-terminal activation domain of
c-Jun on Ser63 and Ser73, causing increased
c-Jun transcriptional activity (17). Results from cotransfection
studies applying pIL6luc(-122) or p(NF-B)3xCAT, both containing
binding sites for NF-B only, in conjunction with pCMV-TAM67, the
NH2-terminally truncated dominant negative c-Jun, suggest
c-Jun/NF-B protein/protein interaction. pCMV-TAM67 specifically
binds to leucine zippers of endogenous Jun or Fos family proteins,
forming low activity AP-1 complexes (51). pCMV-TAM67 binding would be
expected to render the c-Jun and the c-Fos bZip domains unavailable to
p65, which requires the bZip domain for interaction. Alternatively,
pCMV-TAM67 may interact via its bZip domain with p65, rendering the p65
unavailable to interact with c-Jun or c-Fos. In our experiments
pCMV-TAM67 dramatically impaired B-driven promoter activity,
strongly suggesting that either c-Jun or c-Fos may contribute to the
transactivation capacity of NF-B. This finding is in agreement with
a previous report that cross-interaction of c-Jun or c-Fos with the
NF-B subunit p65 leads to synergized potential of NF-B
transactivation (37). Furthermore, in a human keratinocyte progression
model TAM67 was also found to inhibit the activation of NF-B (52).
Taking into account that 1) the JNK pathway affects transcriptional
activity of NF-B, and 2) JNK is the main activator of c-Jun, it is
tempting to speculate that interaction of NF-B with the c-Jun
protein results in maximal transactivation. Since serine kinase
activity is required for the activation of c-Jun by JNK, the inhibitory
effect mediated by H7 could be the consequence of impaired c-Jun
phosphorylation by JNK. In concordance, Berra and co-workers
demonstrated that protein kinase C
, a downstream substrate of Ras,
triggers the activation of a number of kinases and suggested that MEK1
and ERK1/2 may also participate in NF-B activation by enhancing the
AP-1/NF-B cross-coupling mechanism (53). In contrast, a recent study
by Min et al. showed that transient overexpression of an
N-terminal-truncated c-Jun or a catalytically inactive JNK inhibited
TNF-induced transcription of the E-selectin but not of a B-promoter
reporter gene in endothelial cells (54). Thus, the observation that
c-Jun augments B-dependent transcription may be cell type and
stimulus specific.
We cannot exclude that in our cell system, in addition to the ERK and
JNK pathways, the p38 signaling route may also be involved in NF-B
activation. In studies by vanden Berghe et al. TNF-induced
NF-B-mediated transcription, but not NF-B DNA binding, appeared
dependent on activation of the p38/RK pathway in the mouse fibrosarcoma
cell line L929sA (55). However, the signaling modules involved in IL-6
gene regulation are not only cell type specific, but also depend on the
stimulatory signal. More detailed study is required to elucidate the
role of the additional MAPK pathways in IL-6 gene expression.
Until recently limited data existed regarding the way activated kinases might be dephosphorylated and inactivated. Since the MAP kinases ERK1/ERK2 and JNK are involved in the regulation of IL-6 expression, the balance of MAPK kinase and MAPK phosphatase activities determines the state of activation of monocytes. Three mammalian MAPK phosphatases have been identified until now, CL100, PAC-1, and Pyst-1 (56, 57, 58, 59). Although their occurrence in monocytes and leukemic cell lines and the substrate specificity of these PPases have not yet been resolved, they may have important functional roles in mediating cytokine gene expression in monocytes.
We conclude that OA-induced IL-6 gene activation in monocytes is
mediated through activation of the JNK and ERK1/2 pathways. The ERK1/2
pathway was shown to be specifically involved in enhancing the
transactivational capacity of NF-B. Both pathways may provide for
serine kinase activity and c-Jun activation, which were shown to
enhance B-driven reporter gene expression in human monocytes.
|
Footnotes |
|---|
2 Present address: Akzo Nobel, N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands.
3 Address correspondence and reprint requests to Dr. Edo Vellenga, Division of Hematology, Department of Medicine, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail address:
4 Abbreviations used in this paper: AP-1, activator protein-1; NF-IL6, nuclear factor IL-6; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IKK, IB kinase; PPase, protein phosphatase; OA, okadaic acid; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electromobility shift assay.
Received for publication July 13, 1998. Accepted for publication January 22, 1999.
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