From the Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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ABSTRACT |
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The involvement of interleukin (IL)-6 in the
pathogenesis of rheumatoid arthritis (RA) has been recently
demonstrated. IL-1 stimulated rheumatoid fibroblast-like
synoviocytes (FLSs) to produce IL-6 in a concentration- and
time-dependent manner. In the present study we investigated
how the IL-6 promoter is transcriptionally regulated in rheumatoid FLSs
in response to a physiologically relevant mediator of inflammation,
IL-1
. Deletion analysis showed that the IL-6 promoter is regulated
by two positive elements (located at
159 to
142 base pairs (bp) and
77 to
59 bp). Electrophoretic mobility shift assays revealed that
CCAAT/enhancer binding protein-
(C/EBP
) binding to nucleotides
159 to
142 bp was constitutively present. The probe corresponding
to nucleotides
77 to
59 bp gave three positive bands. The two
slower migrating bands were induced by IL-1
and comprised an nuclear
factor (NF)-
B p50/p65 heterodimer and a p65/p65 homodimer. The
faster migrating band was constitutively expressed and identified as
Epstein-Barr virus C-promoter binding factor 1, CBF1. Site-specific
mutagenesis analysis demonstrated that the NF-
B and CBF1 binding
elements regulated inducible activity of the IL-6 promoter in response
to IL-1
stimulation, whereas the C/EBP
binding element mainly
regulated basal activity. We also provide the first evidence that CBF1
functions as a positive regulator of human IL-6 gene transcription.
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INTRODUCTION |
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Rheumatoid arthritis
(RA)1 is a chronic
inflammatory disease in which the synovial environment is characterized
by intense immunological activity (1). In particular, the
macrophage-like synoviocytes and fibroblast-like synoviocytes (FLSs) of
the hyperplastic lining layer exhibit an activated phenotype (2-4).
These cells are a major source of several inflammatory cytokines such
as IL-1, tumor necrosis factor-, and IL-6, proteins which play
crucial roles in the pathophysiology of RA (2, 5, 6).
IL-6 is a pleiotropic cytokine involved in T cell growth (7), B cell differentiation that might lead to the production of rheumatoid factors (8, 9), and the induction of acute phase proteins (10). Immunological disorders often associated with RA include polyclonal plasmacytosis, production of autoantibodies, increased levels of acute phase proteins, and an increased number of peripheral blood platelets, all of which are related to the biological actions of IL-6 (11). In fact, IL-6 is produced mainly by FLSs in the synovium (5, 9) and is found at high levels in the synovial fluids and the serum obtained from RA patients (12, 13). Recently, administration of anti-IL-6 receptor antibody dramatically improved the symptoms and clinical markers of RA patients (14), indicating that IL-6 is one of the key cytokines for the development of RA.
So far, several regulatory elements such as AP-1, multiple responsive
element (MRE), 5'-C/EBP, 3'-C/EBP
, and NF-
B have been
identified within the human IL-6 promoter region (see Fig. 2) (11).
These promoter elements are regulated in a cell type-specific manner.
AP-1, MRE, 5'-C/EBP
, and NF-
B binding elements are required for a
murine monocyte/macrophage cell line, the PU5-1.8 (15), whereas
5'-C/EBP
, 3'-C/EBP
, and NF-
B binding elements are required for
a human monocytic leukemia cell line, the THP-1 cell (16). The NF-
B
site is sufficient for the full expression of this promoter by a human
T cell leukemia virus type I-infected T cell line (17). More recently,
it was reported that Epstein-Barr virus C-promoter binding factor 1 (CBF1) acts as a negative regulator of the IL-6 gene in a mouse
embryonal carcinoma, the F9 cell, and in a mouse fibrosarcoma cell, the
L929sA (18, 19). CBF1 is a DNA-binding protein that is targeted by the
viral transactivator Epstein-Barr virus nuclear antigen 2 in
Epstein-Barr virus-infected human B lymphocytes (20). CBF1 is also
known as recombination binding protein J
involved in the
rearrangement of the immunoglobulin V(D)J gene (21), although
conflicting data were reported (22).
IL-1 is a potent activator of IL-6 synthesis by human rheumatoid
FLSs (9). Although several transcriptional elements have been
implicated in the induction of human IL-6 gene by IL-1
, it is now
apparent that the regulatory mechanisms of human IL-6 gene
transcription are more complex and divergent among various cell types
and phenotypes than initially described (11). There have been no
previous reports regarding the molecular mechanisms of basal
transcriptional activity of the IL-6 gene in the absence of
inflammatory stimulation. In addition, transcriptional regulation of
this gene in rheumatoid FLSs has not been elucidated. In the present
study we investigated how the IL-6 promoter is regulated in rheumatoid
FLSs both, in the unstimulated state and in response to
IL-1
.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Recombinant human IL-1 and IL-6 were obtained
from Genzyme Corp. (Cambridge, MA). Polyclonal antibodies against p50
(sc-114X), p52 (sc-848X), p65 (sc-109X), c-Rel (sc-70X), HMG I(Y)
(sc-1564), C/EBP
(sc-736), and STAT-1 (sc-346) were purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Cells-- Synovial tissue samples were obtained from patients with RA or osteoarthritis (OA) undergoing total joint replacement. All RA and OA patients were evaluated by a certified rheumatologist and were diagnosed as having RA and OA, respectively, according to the criteria of the American College of Rheumatology (23, 24). Written informed consent was obtained from each patient. Each tissue specimen was minced and then digested with 4 mg/ml collagenase for 2 h at 37 °C. Cells were plated in RPMI 1640 (Nikken, Kyoto, Japan) with 10% fetal bovine serum (Life Technologies, Inc.). When the cells had grown to confluence, they were treated with trypsin/EDTA and split at a 1:4 ratio. For experiments, FLSs were used in passages 3-10. A FLS line, MH7A,2 was established from synoviocyte cultures obtained from a 53-year-old female RA patient. The MH7A cells were plated in RPMI 1640 (Nikken, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). When the cells had grown to confluence, they were treated with trypsin/EDTA and split at a 1:5 ratio.
Measurement of IL-6-- Cells at confluence were cultured in RPMI 1640 medium supplemented with 0.1% bovine serum albumin for the designated time periods. The culture supernatants were kept frozen until measured for IL-6, which was done by a specific sandwich enzyme-linked immunosorbent assay. Briefly, supernatants or serial dilutions of recombinant IL-6 standards (Genzyme) were incubated overnight at 4 °C in 96-well microtiter plates (Nunc, Roskilde, Denmark) previously coated overnight at 4 °C with anti-human-IL-6 monoclonal antibody (2 µg/ml; R&D Systems, Minneapolis, MN) and then for 2 h at room temperature for saturation. After the plates had been washed, biotinylated anti-human IL-6 polyclonal antibody (2 µg/ml; R&D) was added. The incubation was carried out for 4 h at room temperature. After subsequent incubation (2 h, room temperature) with horseradish peroxidase-conjugated avidin (Zymed Laboratories Inc., South San Francisco, CA), 3,3',5,5'-tetramethylbenzidine (Dojindo Labs., Kumamoto, Japan) was added to the wells. The absorbance at 450 nm was measured by a microplate reader (Bio-Rad). The minimum detection limit of the assays was 6.25 pg/ml.
Plasmid Construction--
For generation of plasmid pIL6-3BLuc,
a SacI/XhoI fragment containing the 1169-bp
(1158 to +11 relative to the transcription initiation site)
BamHI/XhoI 5'-upstream sequence of the IL-6 gene was excised from the plasmid pGEMhIL-6 GT (Riken Gene Bank, Tsukuba, Japan) (29) and inserted into the compatible site
(SacI/XhoI) of the luciferase reporter plasmid
pGL3-Basic (Promega Corp., Madison, WI). A series of deletion mutants
of the 5'-flanking region of the IL-6 gene were created as follows. For
the construction of pIL6NX-3BLuc, pIL6-3BLuc was digested with
NheI and XhoI to generate the
NheI/XhoI (
225 to +11) fragment of the IL-6
upstream region, which was then cloned into pGL3-Basic. For the
construction of pIL6BX-3BLuc, pIL6AX-3BLuc, pIL6MX-3BLuc, pIL6HX-3BLuc,
and pIL6SsX-3BLuc, pIL6B-3BLuc was digested with BfaI,
AatII, MseI, HaeIII, or
SspI, respectively, made blunt-ended with Klenow enzyme or
T4 polymerase, and released by XhoI digestion. The resulting fragments were subcloned into pBluescript® II KS(+) phagemid vector (Stratagene, La Jolla, CA) which was then digested with SacI
and XhoI. The SacI/XhoI fragment was
next inserted into pGL3-Basic. For the construction of pIL6KX-3BLuc, a
synthesized NheI/XhoI (
77 to +11) fragment of
the IL-6 gene was cloned into the pGL3-Basic.
Transient Transfection and Luciferase Assay--
Cells were
grown to confluence in 75-cm2 culture flasks containing
RPMI 1640 medium supplemented with 10% fetal bovine serum, harvested,
and resuspended in RPMI 1640 medium without phenol red to give a cell
concentration of 2 × 106 cells/ml. A 500-µl volume
of cell suspension, 5-10 µg of the luciferase reporter plasmids, and
0.4 µg of pCMV--galactosidase were placed into a Bio-Rad 4-mm
cuvette; electroporation (Bio-Rad Gene Pulser, 0.29 kV, 960 µF) was
then performed. The transfected cells were cultured for the designated
time periods for each reporter plasmid in RPMI 1640 medium without
phenol red. All assays were carried out in 96-well plates (Culture
PlateTM; Packard Instrument Company, Meriden, CT). The
luminescence was measured with a Top Count (Packard) at the conditions
of 0.15 min of counting time, a single photon counting mode, and a
15-min period of dark adaptation at 22 °C (Top Count). The
-galactosidase activity was measured as described previously (26).
Briefly, cells were washed with PBS(
), and then 100 µl of 1.5 mM chlorophenol red-
-D-galactopyranoside
(Boehringer Mannheim, Germany) in PBS(
) containing 20 mM
KCl, 2 mM MgSO4, 100 mM
2-mercaptoethanol, and 0.5% Nonidet P-40 (Wako) were added. The
absorbance at 570 nm was thereafter measured by a microplate reader
(Bio-Rad).
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared by a method previously reported (27) with slight
modifications. The cells were washed with ice-cold PBS(), harvested,
and resuspended in 400 µl of hypotonic buffer A (10 mM
HEPES (pH 7.9), 10 mM KCl, 10 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) for
15 min on ice. The cells were then lysed by the addition of 0.6%
Nonidet P-40 and vortexing for 10 s. Nuclei were separated from
the cytosol by centrifugation at 12,000 × g for 30 s, washed with 400 µl of buffer A containing 0.6% Nonidet
P-40, resuspended in buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), vigorously vortexed for 15 s, and incubated for 5 min on ice. This step was repeated three times. Nuclear extracts were then
obtained by centrifugation at 12,000 × g for 10 min.
Protein concentration was measured essentially following the method of Bradford (28) using a protein dye reagent (Bio-Rad).
Electrophoretic Mobility Shift Assays (EMSAs)--
Eight
oligonucleotides as shown in Fig. 3 were synthesized with a DNA
synthesizer (Sawady Technology, Tokyo, Japan). The 3' ends of the
oligonucleotides were 32P-labeled with Klenow DNA
polymerase (Megaprime DNA labeling systems; Amersham International plc,
Buckinghamshire, UK). Samples containing 5 µg of nuclear extract were
incubated with 10,000 cpm of labeled oligonucleotides and 1 µg of
poly(dI-dC) in a 10-µl volume of 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 5% glycerol in the presence or absence of competitor
oligonucleotides for 15 min at room temperature and run on 4%
polyacrylamide gels (acrylamide:bisacrylamide, 30:1, wt/wt) in 0.5×
TBE buffer (1× TBE: 89 mmol/liter Tris-HCl, (pH 8.0), 89 mmol/liter
boric acid, and 2 mmol/liter EDTA) at 150 V. The gel was subsequently
dried and exposed to Rx-U film (Fuji Photo Film, Tokyo, Japan) at
70 °C.
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RESULTS |
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Induction of IL-6 by IL-1 in Rheumatoid FLSs and OA
FLSs--
Initial studies were designed to confirm the induction of
IL-6 synthesis by IL-1
in the human rheumatoid FLSs. Rheumatoid MH7A
cells, primary rheumatoid FLSs, and primary OA FLSs were incubated with
various concentrations of IL-1
for 24 h. For all cells there
was a concentration-dependent increase in the amount of
IL-6 released into the medium (Fig.
1A). Peak IL-6 production was
noted with 1-10 ng/ml IL-1
. After the addition of 1 ng/ml IL-1
,
IL-6 production was detectable within 2 h and reached a maximum at
24 h (Fig. 1B). IL-6 production by the FLSs of RA
patients in response to IL-1
was significantly greater than that by
the control FLSs from patients with OA (Fig. 1).
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Functional Analysis of 5'-Cis-regulatory Elements of the Human IL-6
Gene Operating in Rheumatoid MH7A Cells and OA FLSs--
As
illustrated in Fig. 2, approximately 1200 bp of the 5'-flanking region of the human IL-6 gene contains various
putative responsive elements: AP-1 (283 to
277), MRE (
168 to
153), 5'-C/EBP
(
155 to
148), 3'-C/EBP
(
83 to
75),
IL6-
B (
73 to
60), and TATA box (
53 to
47 and
30 to
23)
(11, 15, 29, 30). Functional analysis of the 5'-flanking region of the
human IL-6 gene was carried out using the
1158 to +11 fragment as
well as a series of 5'-deletion mutants of the IL-6 promoter linked to
the luciferase reporter gene (Fig. 2). For transient expression of the
reporter gene, each plasmid was used for transfection of rheumatoid
MH7A cells and OA FLSs by electroporation, and the luciferase activity
of the cell lysates was then measured 18 h after the addition of
IL-1
(1 ng/ml). IL-1
markedly induced the luciferase activity in
the MH7A cells transfected with the luciferase reporter plasmid
carrying the IL-6 promoter (Fig. 2A). The luciferase
activity significantly decreased when two regions (
159 to
142 bp
and
77 to
59 bp) were deleted. The C/EBP
consensus element
(
155 to
148 bp), NF-
B consensus element (
73 to
64 bp), and
CBF1 consensus element (
67 to
60 bp) were contained in these two
gene segments. 5'-CATGGGAA-3' (
60 to
67) was homologous to the
reported CBF1 consensus binding element 5'-A(G/C)CGTGGGAA-3' (31).
IL-1
induced less luciferase activity in the OA FLSs transfected
with the luciferase reporter plasmids carrying the IL-6 promoter
compared with that induced in the rheumatoid FLSs (Fig.
2B).
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EMSA Targeting Positive Regulatory Elements--
For the further
identification of these positive regulatory elements, we prepared eight
double-stranded oligonucleotides as shown in Fig.
3. C/EBP (
158 to
142)
oligonucleotide contained the C/EBP
binding element, and IL6-
B
(
75 to
60) oligonucleotide contained the NF-
B and CBF1 binding
elements. C/EBP
-mt was the mutant oligonucleotide for C/EBP
.
IL6-
B-mt1 was the mutant for NF-
B. IL6-
B-mt2 was the double
mutant for NF-
B and CBF1. IL6-
B-mt3 was the mutant for CBF1.
Ig-
B oligonucleotide was the NF-
B consensus binding element of
the mouse immunoglobulin
chain gene (32). The m8 oligonucleotide
contained the CBF1 consensus binding element of the m8 gene of
Drosophila Enhancer of split (31). We carried out EMSA using
the nuclear extracts of MH7A cells that had been either unstimulated or
stimulated with IL-1
for 4 h. As shown in Fig.
4, when the C/EBP
oligonucleotide was
used as a probe, a broad band was constitutively detected (lanes
1 and 2). This complex was specifically competed by the
unlabeled C/EBP
oligonucleotide (lanes 3 and
4). The complex was scarcely observed when the C/EBP
-mt oligonucleotide containing a 5-bp mutation of the C/EBP
element was
used as a probe (lane 5). This band disappeared with the
addition of anti-C/EBP
antibody (lane 8).
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Separation of NF-B and CBF1 Bindings by the Site-specific
Mutagenesis of the IL6-
B Element--
EMSA using the IL6-
B
oligonucleotide showed the complex nature of the DNA-protein
interactions at this motif involving proteins of the NF-
B p50/p65
heterodimer, p65/p65 homodimer, and CBF1 (Fig. 5). So we introduced
site-specific mutations into this motif to separate CBF1 binding from
NF-
B binding. The effects of the mutations on DNA-protein
interactions were then determined by EMSA. Fig.
6 shows that the IL6-
B-mt1
oligonucleotide, which contained a 3-bp mutation in the NF-
B binding
element (Fig. 3), enhanced CBF1 binding but eliminated NF-
B binding
(lane 3). Competition with the unlabeled IL6-
B-mt1
oligonucleotide eliminated CBF1 binding complex of the unstimulated
(data not shown) and the stimulated (lanes 8 and
9) nuclear extracts, but did not affect the binding of
NF-
B (lanes 8 and 9). The IL6-
B-mt2
oligonucleotide containing a 4-bp mutation in both NF-
B and CBF1
binding elements (Fig. 3) eliminated all specific binding (lane
4). Competition with the unlabeled IL6-
B-mt2 oligonucleotide
did not affect either NF-
B or CBF1 binding (lanes 11 and
12). The IL6-
B-mt3 oligonucleotide containing a 2-bp
mutation in the CBF1 element (Fig. 3) permitted NF-
B binding, but
eliminated CBF1 binding (lane 5). Competition with the
unlabeled IL6-
B-mt3 oligonucleotide eliminated the binding of
NF-
B (lanes 14 and 15). The Ig-
B
oligonucleotide did not bind CBF1 protein (lane 6), as
expected from the competition experiment using the unlabeled Ig-
B
oligonucleotide (Fig. 5B, lanes 6 and 7).
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Cooperation of NF-B and CBF1 Is Required for IL-1
-induced
Gene Transcription--
For analysis of the functional significance of
NF-
B and CBF1 bindings, luciferase reporter constructs containing
four copies of the NF-
B and/or CBF1 binding elements connected to
the tk promoter gene were constructed (illustrated in Fig.
7A) and used to transfect
rheumatoid MH7A cells, primary rheumatoid FLSs, and primary OA FLSs.
Luciferase activity was measured 6 h after IL-1
stimulation. A
representative result is shown in Fig. 7B. The reporter
construct carrying both NF-
B and CBF1 binding elements (IL6-
B × 4) was clearly transcribed in response to IL-1
. In contrast, IL6-
B-mt1 × 4, IL6-
B-mt3 × 4, and
Ig-
B × 4 reporter constructs were not significantly
transcribed by rheumatoid MH7A cells or primary rheumatoid FLSs in
response to IL-1
. In this regard, the Ig-
B × 4 reporter
construct was transcribed by Jurkat T cells stimulated with
phytohemagglutinin and phorbol 12-myristate 13-acetate (data not
shown). The IL6-
B × 4 reporter construct was significantly
less transcribed by OA FLSs in response to IL-1
compared with that
transcribed by rheumatoid FLSs. There was no significant difference in
the luciferase activity among OA FLSs transfected with IL6-
B × 4, IL6-
B-mt3 × 4, or Ig-
B × 4 reporter constructs.
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Requirement of C/EBP, NF-
B, and CBF1 for IL-6 Gene Activation
by IL-1
--
For determination of the functional role of each DNA
binding element in IL-6 gene transcription by rheumatoid FLSs and
normal FLSs, the IL-6 promoter containing mutations in the NF-
B,
NF-
B plus CBF1, or CBF1 binding element was used for transfection of rheumatoid MH7A cells and OA FLSs. A representative result is shown in
Fig. 8. In rheumatoid MH7A cells (Fig.
8A), the activity of the wild-type IL-6 promoter was
increased by 4.6-fold upon stimulation with IL-1
. A 5-bp mutation
introduced into the C/EBP
binding element reduced the basal IL-6
promoter activity to 51% of the wild-type value, and reduced the
induction of IL-6 promoter in response to IL-1
to 40%. A 3-bp
mutation introduced into the NF-
B element of the IL-6 promoter did
not affect the basal activity of this promoter, but reduced the
IL-1
-inducible activity to 74%. A 2-bp mutation introduced into the
CBF1 binding element also reduced the IL-1
-inducible activity to
68%, whereas it did not affect the basal activity. Mutations in both
NF-
B and CBF1 binding elements did not affect the basal activity,
but totally abolished the induction of this promoter activity by
IL-1
. These results demonstrate that the C/EBP
binding element is
required for the full expression of the IL-6 promoter in rheumatoid
MH7A cells, although both NF-
B and CBF1 binding elements are
critical for the transcriptional induction in response to IL-1
. In
contrast to rheumatoid FLSs, OA FLSs exhibited significantly lower
luciferase activity upon stimulation with IL-1
(Fig. 8B).
The C/EBP
mutation reduced the basal IL-6 promoter activity of OA
FLSs to 46% of the wild-type value, and reduced the induction of IL-6
promoter in response to IL-1
to 39%. The NF-
B mutation of the
IL-6 promoter did not affect the basal activity, but completely
abolished the IL-1
-inducible activity. The CBF1 mutation did not
affect either the basal or the IL-1
-inducible activity of the IL-6
promoter in OA FLSs.
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DISCUSSION |
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Our present study clearly demonstrated that IL-1 stimulated
rheumatoid FLSs to produce IL-6 in a concentration- and
time-dependent manner (Fig. 1). IL-6 production of
rheumatoid FLSs was significantly enhanced compared with that of OA
FLSs. IL-1
-induced up-regulation of IL-6 promoter activity was
localized to two DNA segments, positions
159 to
142 bp and
77 to
59 bp, by the deletion analysis (Fig. 2). The former positive motif
was identified as the C/EBP
binding site, and the latter was the
complex binding sites of the NF-
B p50/p65 heterodimer, p65/p65
homodimer, and CBF1 (Figs. 3-5). The NF-
B and CBF1 binding elements
regulated inducible IL-6 promoter activity in response to IL-1
in
rheumatoid FLSs, whereas the C/EBP
binding element mainly influenced
basal activity (Figs. 2, 7, and 8). In addition we have provided the
first evidence that CBF1 functions as a positive regulator of human
IL-6 gene transcription in rheumatoid FLSs (Figs. 7 and 8). In contrast to that by rheumatoid FLSs, induction of IL-6 promoter activity by OA
FLSs was less pronounced (Fig. 2B). CBF1 seemed not to be involved in IL-1
-induced up-regulation of the IL-6 promoter by OA
FLSs (Figs. 7 and 8).
RA is a chronic inflammatory disease characterized by the proliferation
of the synovial membrane into a highly vascularized tissue known as a
pannus. The pannus consists of several distinct cell types, which
include resident FLSs and infiltrating mononuclear cells capable of
producing imflammatory cytokines such as IL-1, tumor necrosis
factor-, and IL-6 (33). Several studies including ours have
demonstrated that rheumatoid FLSs produce large amounts of IL-6 upon
stimulation with IL-1 or tumor necrosis factor-
(9). IL-6 induces
the production of acute phase proteins by hepatocytes (34). It also
facilitates differentiation of B cells and may contribute to the
production of rheumatoid factors (11). Moreover, IL-6 is involved in
Fc
RI expression on monocytes through the induction of STAT family
factors (35). The excessive production of IL-6 seems to be related to
the immunological abnormalities associated with RA. Therefore, it is
essential to delineate the molecular and cellular mechanisms of IL-6
production by rheumatoid FLSs. Rheumatoid FLSs, however, pose several
problems as useful in vitro models, including their limited
source and growth potential, requirement for a high concentration of
serum for growth, and lot-to-lot variability in functional assays.
Recently, we established a rheumatoid FLS line, MH7A,2
derived from an RA patient, and showed herein that the MH7A cells functionally retained IL-1
-inducible IL-6 production (Fig. 1) and
transcriptional inducibility of the IL-6 promoter and 4 tandem repeats
of the IL6-
B element in response to IL-1
(Figs. 2, 7, and 8).
With this system we sought to elucidate the transcriptional regulation
of the human IL-6 gene by rheumatoid FLSs.
Transcriptional regulation of the IL-6 promoter involves the
interaction of transcription factors such as NF-B/rel family, C/EBP
of the bZIP family, cAMP-response element binding protein, and AP-1
(11). As different sets of transcription factors may regulate the IL-6
gene in a cell type-specific manner, it is still uncertain which
element is functional in the rheumatoid FLSs. Therefore, we first
performed deletion analysis of the IL-6 promoter and found that the
IL6-
B motif was critical for the transcriptional induction of the
IL-6 promoter in IL-1
-stimulated rheumatoid MH7A cells and OA FLSs
and that the 5'-C/EBP
element was further required for the full
transcriptional activation of this promoter (Fig. 2). The IL6-
B
motif seemed to function more efficiently for the transcriptional
induction in rheumatoid FLSs than in OA FLSs.
Next, we performed EMSAs to identify the transcription factors binding
to these elements of the IL-6 promoter. NF-B binding activity was
significantly up-regulated in rheumatoid MH7A cells stimulated with
IL-1
(Fig. 5A, lane 2). Supershift experiments identified the two slower IL6-
B binding complexes to contain NF-
B
p50/p65 heterodimer and p65/p65 homodimer (Fig. 5B). It was
reported that the p65 subunit of NF-
B is responsible for the
transactivation due to the existence of a potent transactivation domain
located in its C-terminal portion (36-38). It is well assumed that
IL-1
-induced up-regulation of the p65 subunit may lead to transcriptional induction by IL-6 promoter in rheumatoid FLSs. In fact,
several investigators identified the existence of NF-
B p50 and p65
subunits in the nuclei of cells located in the lining and subsynovial
regions of the rheumatoid synovium in situ (39, 40).
CBF1 was identified as a constitutive component of the IL6-B binding
complex (Fig. 5B). The mutation introduced in the CBF1 binding element significantly reduced the response to IL-1
(Fig. 8A), indicating the accessory role of this element in
rheumatoid FLSs. A mutation introduced into the CBF1 binding element
resulted in a profound loss of the transcriptional induction of 4 tandem repeats of IL6-
B element in response to IL-1
in rheumatoid
FLSs, but not in OA FLSs (Fig. 7). Our data clearly indicated that CBF1 acted as a positive regulator of the human IL-6 gene transcription by
rheumatoid FLSs. It has been reported that CBF1 functions as a negative
regulator of the IL-6 gene in mouse cell lines (18, 19). The
differences in species and cell types may account for the discrepancy
regarding the opposite CBF1 functions. In addition, the double mutant
of NF-
B and CBF1 binding elements totally abolished the
transcriptional induction of IL-6 promoter (Fig. 8A),
suggesting that the cooperation of the two elements of the IL-6
promoter plays an essential role in the efficient transcriptional
induction of the IL-6 gene in response to IL-1
in rheumatoid FLSs.
The finding is consistent with the report that the constitutively activated form of human Notch1 binds to CBF1 and activates
transcription through the CBF1-responsive element (41). In this regard,
HMG I(Y) was previously shown to be required for virus induction of the
interferon-
gene (42). HMG I(Y) is unable to stimulate (or inhibit)
promoter activities by itself but is able to interact with NF-
B to
modify promoter activity (43). CBF1 might behave by itself like HMG
I(Y), whereas HMG I(Y) was not involved in the transcriptional
regulation of the IL6-
B motif.
In contrast to the inducible binding of NF-B, C/EBP
binding
activity was constitutively present in rheumatoid MH7A cells (Fig. 4).
Deletion of this element or 5-bp mutants revealed that C/EBP
was
involved in the basal level of transcription rather than in the
induction of the IL-6 promoter (Figs. 2 and 8, A and B). It is interesting to note, however, that the induction
of transcriptional activity was reduced in proportion to the basal activity when the C/EBP
element was deleted or mutated (Figs. 2 and
8, A and B). The C/EBP
binding element seems
essential for the full activation of this promoter in both rheumatoid
and OA FLSs. Several investigators have suggested that physical
interaction between C/EBP
and NF-
B lead to synergistic
transactivation of the IL-6 promoter through the 5' C/EBP
, 3'
C/EBP
, and NF-
B sites (16) in a way similarly observed for the
human IL-8 promoter (44). Although the IL-6 promoter activity with the
double mutation of NF-
B and CBF1 binding elements was not induced by
IL-1
, the 5'-C/EBP
element along with either NF-
B or CBF1
element was sufficient for the activation by IL-1
(Fig. 8,
A and B), indicating that the physical
interaction of C/EBP
with either NF-
B or CBF1 synergistically
transactivates the IL-6 promoter through 5'-C/EBP
and IL6-
B
motifs.
IL-1 signaling is initiated by its type I receptor, which triggers the
mitogen-activated protein (MAP) kinase cascade, leading not only to
IB phosphorylation followed by the translocation of NF-
B
complexes into the nucleus (45), but also to phosphorylation and
transactivation of C/EBP
(46). The MAP kinase cascade is now
classified into three different types of MAP kinase systems, i.e. p42/p44, p54/JNK, and p38/RK (47). More recently, it
has been reported that a specific p38 MAP kinase inhibitor, SB203580, significantly inhibited the IL-6 production by MRC-5 fibroblasts (48);
whereas a selective inhibitor of the p42/p44 MAP kinase pathway,
PD098059 (49), had no effect on the IL-6 production by MH7A
cells.2 Our present study demonstrated that human
rheumatoid FLSs produced larger amounts of IL-6 upon stimulation with
IL-1
compared with OA FLSs. The mutation introduced in the NF-
B
binding element reduced the response to IL-1
by no more than 30% in
rheumatoid FLSs, but totally abolished the response to IL-1
in OA
FLSs (Fig. 8B) and other cell types (50), indicating that
IL-6 gene regulation by the IL6-
B element is altered in rheumatoid
FLSs. The CBF1 element was shown to be involved in the transcriptional
induction by rheumatoid FLSs but not in that by OA FLSs (Figs. 7 and 8, A and B). Thorough elucidation of the
transcription factors and elements involved in the IL-6 gene
transcription in rheumatoid FLSs might facilitate the future
development of a specific IL-6 synthesis inhibitor for RA therapy.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tasuku Honjo (Kyoto University) for providing the anti-CBF1 antibody and Dr. Larry D. Frye for reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from Kissei Pharmaceutical Co., Ltd.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: Kissei Pharmaceutical
Co., Ltd., 4365-1 Kashiwabara, Hotaka Minamiazumi, Nagano 399-8304, Japan. Tel.: 81-263-82-8820; Fax: 81-263-82-8826.
1
The abbreviations used are: RA, rheumatoid
arthritis; FLS, fibroblast-like synoviocyte; IL, interleukin; Luc,
luciferase; tk, thymidine kinase; mt, mutant; CMV, cytomegalovirus; NF,
nuclear factor; C/EBP, CCAAT/enhancer-binding protein; MRE, multiple
response element; AP-1, activated protein-1; bp, base pair(s); EMSA,
electrophoretic mobility shift assay; PBS(), phosphate-buffered
saline without Ca2+ and Mg2+; CBF1, C-promoter
binding factor; OA, osteoarthritis; MAP, mitogen-activated protein;
STAT, signal transducer and activator of transcription.
2 K. Miyazawa, A. Mori, K. Yamamoto, and H. Okudaira, manuscript in preparation.
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REFERENCES |
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