From the Laboratoire de Biochimie du Tissu
Conjonctif, Faculté de Médecine, 14032 Caen Cedex, France
and the ¶ Department of Veterinary Basic Sciences, Royal
Veterinary College, Royal College Street,
London NW1 OTU, United Kingdom
Received for publication, November 9, 2001, and in revised form, October 29, 2002
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ABSTRACT |
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Signal transducers and activators of
transcription (STAT) factors are cytoplasmic proteins that can be
activated by Janus kinases (JAK) and that modulate gene expression in
response to cytokine receptor stimulation. STAT proteins dimerize,
translocate into the nucleus, and activate specific target genes. In
the present study, we show for the first time that interleukin-6 (IL),
in the presence of its soluble receptor (sIL-6R), induces activation of
JAK1, JAK2, and STAT1/STAT3 proteins in bovine articular chondrocytes. Western blotting and mobility shift assays demonstrated that this effect is accompanied by the DNA binding of the STAT proteins. The
mitogen-activated protein kinase pathway was also activated in response
to IL-6/sIL-6R association, as reflected by phosphorylation of ERK1 and
ERK2 proteins. In these conditions, the expression of
cartilage-specific matrix genes, type II collagen, aggrecan core, and
link proteins was found to be markedly down-regulated. This negative
effect was abolished by addition of parthenolide, an inhibitor
of the STAT activation, whereas blockade of the MAP kinases with
PD098059 was without significant effect. Thus, activation of the STAT
signaling pathways, but not ERK-dependent pathways, is
essential for down-regulation of the major cartilage-specific matrix
genes by IL-6. In addition, a parallel reduction of Sox9 expression, a key factor of chondrocyte phenotype, was found in these
experimental conditions. These IL-6 effects might contribute to the
phenotype loss of chondrocytes in joint diseases and the alteration of
articular cartilage associated with this pathology.
Interleukin-6 (IL-6)1 is
a pleiotropic factor that belongs to a cytokine subfamily, whose
members include ciliary neurotrophic factor, leukemia inhibitory
factor, interleukin-11, oncostatin M, and cardiotrophin-1, and which
share a common signal transducing molecule, gp130, in their respective
complexes (1). IL-6 family members have been shown to induce the Janus
kinase (JAK)/signal transducer and activator of transcription (STAT)
signaling pathway in several systems (2). JAKs are cytoplasmic tyrosine
kinase effectors that are activated by ligand-receptor interactions, leading to tyrosine phosphorylation and activation of the cytoplasmic transcription factors, STATs, which then translocate to the nucleus and
act on target gene transcription. Four kinases of the JAK family and
seven mammalian STATs have been identified, which may be activated
individually or in combination (3).
IL-6 initiates its action by binding to its receptor, which is composed
of the 80-kDa ligand-binding subunit (IL-6R) and the signal-transducing
subunit, gp130 (4). After ligand binding, the gp130 subunits aggregate
into a complex consisting of the IL-6R, two gp130 molecules, and one
IL-6 molecule (5). This aggregation activates the gp130-associated
protein-tyrosine kinases JAK1, JAK2, and TYK2, which tyrosine
phosphorylate gp130, themselves, and STAT3 and -1. Tyrosine
phosphorylation of STAT3/1 occurs at a single residue
(STAT3-Tyr705; STAT1-Tyr701) which is located
in the conserved SH2 allowing homo- as well as
heterodimerization (6). During translocation to the nucleus, STAT3 and
STAT1 are specifically phosphorylated on a serine residue, a
prerequisite for being fully active on gene transcription (7). However,
cellular responses to IL-6 can also be implemented via gp130-mediated
signaling through interaction of IL-6 with a soluble form of the IL-6R
(sIL-6R), which is released by either differential IL-6R mRNA
splicing or proteolytic shedding from the cell membrane (review in Ref.
8). This naturally occurring sIL-6R is present in several body fluids
of patients with various diseases as well as in healthy subjects
(9-11). IL-6 regulates several functions, including immunological
reactions in host defense, inflammation, hematopoiesis, and oncogenesis
(12-13). Accumulating evidence suggest that IL-6, as well as the other
IL-6 family member oncostatin, is implicated in both inflammatory and
degenerative joint diseases (rheumatoid arthritis (RA) and
osteoarthritis). IL-6 has been shown to increase the amount of
inflammatory cells in synovial tissue (14) and to amplify the IL-1
effects on the increased metalloprotease synthesis and inhibition of
proteoglycan production (15). The levels of IL-6 in synovial fluid and
serum are significantly higher in RA and osteoarthritis patients
relative to controls (16-20). Furthermore, transgenic mice
overexpressing IL-6 display abnormal features, including symptoms
observed in human RA (21, 22). Today, IL-6 blocking strategy has become
a potential approach to treat the IL-6-related immune inflammatory
diseases of humans such as Castleman's disease and RA (22). Indeed,
inactivation of the IL-6 gene completely protected DBA/1J mice from
collagen-induced arthritis (23) or it delayed the onset of the
pathological process and also reduced its severity (24), indicating
that IL-6 seems to be required for the development of arthritis.
Anti-mouse IL-6R antibody treatment also suppressed the development of
arthritis and protected knee joints from destructive change in DBA/1J
mice immunized with bovine type II collagen (25). Taken together with
the findings that treatment of the collagen-induced arthritis with
anti-tumor necrosis factor- Although IL-6 may contribute to the destructive changes of bone and
cartilage accompanying joint diseases, its mechanism of action on the
cartilage and bone cells and the pathways whereby it controls their
gene expression are unknown. Only a limited number of in
vitro findings have been reported. IL-6 in the presence of soluble
IL-6R has been shown to activate osteoclasts to induce bone resorption
in vitro, suggesting that IL-6 may be involved in
osteoporosis (30). A combination of IL-6 and soluble IL-6R was found to
activate the JAK/STAT and MAP kinase pathways in MG-63 human
osteoblastic cells (31). In articular chondrocytes, contradictory
results have been reported on the effects of IL-6 on proteoglycan
synthesis. It must be noted that in most of the related studies, the
IL-6 effect was investigated in connection with that of other
cytokines, such as IGF-1 and IL-1. For example, very high doses of IL-6
were found to decrease the enhancing effect of IGF-1 on proteoglycan
synthesis (32). It was also shown that IL-6 was required for the
inhibition of proteoglycan synthesis by IL-1 in human articular
chondrocytes (15) but the latter results were not reproduced by others
(33). The contradictory results may be because of the fact that IL-6
effects were investigated in the absence of soluble IL-6 receptor.
Indeed, more recent work from Guerne et al. (34) indicates
that levels of membrane-anchored IL-6 receptor on chondrocytes are
lower compared with those on other cell types, such as hepatocytes, and
that in vitro addition of sIL-6R to IL-6 is required to
observe the full effect of the cytokine. However, until recently, the
IL-6 signaling pathway for these cells has not been identified nor has
the functional significance been investigated on their effects on the
major extracellular matrix components.
The aim of the present study was therefore to analyze the cytoplasmic
signaling pathways that were activated in IL-6/sIL-6R-treated bovine
articular chondrocytes, focusing on the Janus kinases (JAKs) and signal
transducers and activators of transcription (STATs), which are known to
be involved in IL-6 signaling, and the mitogen-activated protein (MAP)
kinase pathway, which may play a role in IL-6 signaling (35-37). In
addition, we determined the relationship between the activation of
these IL-6 signaling pathways and the control of type II collagen,
aggrecan core and link protein gene expression.
The high mobility group box containing transcription factor Sox9 has
been shown to be required for chondrocyte differentiation and for
expression of cartilage-specific marker genes including type II
collagen, collagen IX, collagen XI, and aggrecan (38). Sox9 was found
to bind and activate a specific enhancer element in both the type II
collagen (39) and aggrecan gene (40), and its expression profile was
correlated with that of collagen II during the chondrogenesis process
(41). Therefore, it was interesting to examine Sox9 expression in the
present experimental protocol to investigate the potential implication
of this transcription factor in the IL-6-induced effect on matrix gene
expression by chondrocytes.
Reagents--
Human recombinant IL-6 was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY), human recombinant sIL-6R was
from R & D Systems (Abingdon, NY), and IL-1 Culture and Treatment of Articular Chondrocytes--
Normal
bovine articular cartilage was obtained from the knee joints of freshly
slaughtered calves through a local slaughterhouse and chondrocytes were
cultured as previously described (42). Slices of cartilage were
dissected out and kept in Earle's balanced salt solution. Chondrocytes
were released by digestion with type XIV protease (4 mg/ml) for
1.5 h and type I collagenase (1 mg/ml) overnight in Dulbecco's
modified Eagle's medium at 37 °C. The cells were centrifuged,
washed three times, and seeded at high density (3 × 107 cells/55-cm2 flask) in Dulbecco's modified
Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated
fetal calf serum and antibiotics: penicillin (100 IU/ml), streptomycin
(100 µg/ml), and Fungizone (0.25 µg/ml). The cells were allowed to
recover for 48 h at 37 °C in a humidified atmosphere
supplemented with 5% CO2. Then, cells were serum-starved for 16 h before IL-6 and/or sIL-6R treatments as indicated in the
figure legends. Experiments were performed three times and data from a
representative experiment are shown.
Use of Parthenolide and PD 098059--
In some cases,
parthenolide (Sigma), an inhibitor of phosphorylation and activation of
STAT (43), at 50 µM and PD 098059 (Sigma), an inhibitor
of MAPK pathway (44, 45), at 25 µM were added after serum
deprivation and 2 h before the cytokine treatment. These
inhibitors were dissolved in dimethyl sulfoxide (Me2SO) to
give a concentration of 50 mM, and diluted in Dulbecco's
modified Eagle's medium immediately prior use. Control incubations
contained the same amount of the vehicle. The maximal final
concentration of Me2SO in the cultures was 0.5% (w/v).
Preparation of Cytoplasmic and Nuclear Extracts--
Following
treatment, chondrocytes were rinsed once with ice-cold
phosphate-buffered saline, and lysed in RIPA buffer to prepare cellular
extracts for immunoprecipitation, kinase assays, and for Sox9 Western
blot. RIPA buffer consisted of 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM NaF, 1% Nonidet P-40, leupeptin, pepstatin A, and
aprotinin at 1 µg/ml, and 10 µg/ml phenylmethylsulfonyl fluoride, 1 mM Na3VO4. Cells were scraped off
the plates, incubated on ice for 30 min, and centrifuged at 14,000 × g for 15 min. Alternatively, chondrocytes were also lysed
in hypotonic buffer to prepare nuclear extracts (46, 47). Hypotonic
buffer contained 10 mM Hepes (pH 7.9), 0.1% Nonidet P-40,
1.5 mM MgCl2, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 5 mM NaF, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, and aprotinin at 10 µg/ml, 1 mM
Na3VO4. Hypertonic buffer was composed of 20 mM Hepes (pH 7.6), 25% glycerol, 1 mM
MgCl2, 420 mM NaCl, 0.2 mM EDTA,
0.25 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, and aprotinin at 10 µg/ml, 1 mM Na3VO4. Cellular extracts from
HepG2 cell line were likewise lysed in RIPA buffer and used as positive
controls for TYK2 expression. The protein amount was determined by the
Bradford colorimetric procedure (Bio-Rad).
Immunoprecipitation and Western Blot Analysis--
Cellular
extracts were preincubated with protein A-Sepharose beads to reduce
nonspecific binding. Protein levels were determined on the supernatants
before immunoprecipitation to have equal amounts of immunoprecipitated
proteins. The samples (1-1.5 mg of protein) were incubated with JAK1,
JAK2, or TYK2 antibodies overnight at 4 °C, followed by
immunoprecipitation with protein A-Sepharose beads. Immunoprecipitates
were washed twice with 0.14 M NaCl, 0.01 M
Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.1 mM
Na3VO4, twice with 0.05 M Tris-HCl
(pH 6.8), 0.1 mM Na3VO4, and boiled
for 5 min in Laemmli buffer with 1 mM
Na3VO4 (48). The supernatants were then
analyzed by immunoblotting with JAK1, JAK2, TYK2, or Tyr(P)
antibodies as follows. Immunoprecipitated proteins, cytoplasmic extracts (15-30 µg of protein), or nuclear extracts (20 µg of protein) were subjected to SDS-PAGE under reducing conditions and electrophoretically transferred to polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences, Zawentem,
Belgium). Membranes were blocked for 1 h at room temperature in
Tris-buffered saline (pH 7.6) with 0.1% Tween 20 (TBS-T) and 10%
nonfat dry milk. Then, they were rinsed twice in TBS-T and incubated
overnight at 4 °C with primary antibody. After washing with TBS-T,
they were incubated for 1 h with the appropriate secondary
antibody. The membranes were developed with the ECL+Plus
chemiluminescence detection kit. To check for the presence of equal
amounts of protein or to analyze the expression of several proteins,
blots were stripped (100 mM 2-mercaptoethanol, 2% SDS,
62.5 mM Tris-HCl (pH 6.7)) for 30 min at 50 °C with
stirring, and reprobed.
In Vitro Kinase Assay of MAP Kinase Activity--
MAP kinase
activity was determined using myelin basic protein (MBP) as a
substrate. To 10 µg of cellular extract were added 20 µl of kinase
buffer (25 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 2 mM MnCl2, 1 mM
DTT, 500 µM EDTA, and 40 µM ATP), 1 µg of
MBP, and 1 µCi of [ Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts (7.5 µg of protein) were incubated in binding buffer for 30 min at 25 °C with the cDNA probes, radiolabeled with
[ RNA Extraction and Northern Blot--
Total RNA was extracted
from the chondrocyte cultures by the guanidium
isothiocyanate-phenol-chloroform procedure, with an additional
precipitation step in 6 M LiCl to remove any residual trace
of genomic DNA (49). RNA was fractionated by electrophoresis on 1%
agarose/MOPS/formadelyde gel and transferred to a nylon membrane
(Biodyne B, PerkinElmer Life Sciences). RNA samples were fixed on the
membrane by UV exposure. Type II collagen, aggrecan core and link
protein, and GAPDH probes were generated by reverse transcription-polymerase chain reaction, using the following primers: type II collagen, forward primer, GACCCCATGCAGTACATG; reverse primer,
GACCGTCTTGCCCCACTT; aggrecan core protein, forward primer, CCCTGGACTTTGACAGGGC, reverse primer, AGGAAACTCGTCCTTGTCTCC; link protein, forward primer, CTCACTCTGGAAGATTATGGG, reverse primer, CACAGCATCCTGGTCCAG; GAPDH, forward primer, TGGTATCGTGGAAGGACTCATGAC, reverse primer, ATGCCAGTGAGCTTCCCGTTCAGC.
The probes were 32P-labeled by random priming using the
Strip-EZ DNATM kit (Ambion, Austin, TX). Prehybridization
(1 h) and hybridization (18 h) were performed at 42 °C in
ULTRAhybTM hybridization buffer (Ambion). Blots were washed
several times in 2× SSC (standard saline citrate) and 0.1% SDS at
42 °C. Final washes were in 0.1× SSC plus 0.1% SDS at 42 °C. To
detect several mRNA signals, filters were stripped according to the
manufacturer's instructions. The 32P-labeled
cDNA-mRNA hybrids were visualized by autoradiography.
Reverse Transcription-Polymerase Chain
Reaction--
Two-microgram samples of total RNA were reverse
transcribed into cDNA in a volume of 50 µl containing 10 µl of
5× First Strand buffer, 20 µM oligo(dT)16, 1 µl of
recombinant ribonuclease inhibitor RNAseOUT (40 units/µl), 3 µl of
dNTPs (10 mM each), and 1 µl of Moloney murine leukemia
virus (200 units/µl) (Invitrogen) for 15 min at 42 °C and 5 min at
99 °C. Amplification of generated cDNA was performed in a
thermocycler using the Invitrogen PCR kit, in the presence of both
sense and antisense primers: Sox9, sense
5'-ACAACCCGTCTACACACAGC-3', antisense
5'-ACGATTCTCCATCATCCTCC-3'; The IL-6-soluble Receptor Is Required for JAK1 and JAK2 Tyrosine
Phosphorylation by IL-6 in Articular Chondrocytes--
To examine
whether chondrocytes from normal bovine articular cartilage (BAC)
respond to IL-6, primary cultures of bovine articular chondrocytes kept
in serum-deprived medium for 16 h were subjected to a 10-min
treatment with IL-6 (500 ng/ml), in the presence or absence of sIL-6R
(500 ng/ml). Because IL-6 was previously found to activate JAK1, JAK2,
and TYK2 in several cell types, we wanted to determine whether this JAK
family kinases were also involved in IL-6 signaling in chondrocytes.
Cellular extracts were immunoprecipitated with anti-JAK1, anti-JAK2,
and anti-TYK2 antibodies and immunoblotted with anti-Tyr(P) antibody.
The same membranes were reblotted with JAK1, JAK2, and TYK2 antibodies.
The results revealed that BAC express JAK1 and JAK2 in normal
conditions (Fig. 1, A and
B). In contrast, barely detectable amounts of TYK2 could be
detected in these cells, either in controls (Fig. 1C) or in
IL-6/sIL-6R-treated cultures (not shown). As a control, cellular
extracts from human hepatoma HepG2 cells clearly displayed a
significant level of TYK2, validating the antibody efficiency.
Phosphorylation of JAK1 and JAK2 was only found in the case of
treatment with associated IL-6 and sIL-6R, each element being
separately inactive (Fig. 1, A and B). The dose
of 500 ng/ml for IL-6 and sIL-6R was used here to obtain maximum effect
but lower concentrations were also found to be active (see Fig.
2) and alternatively used for the subsequent study.
IL-6/sIL-6R Induces Dose- and Time-dependent
Phosphorylation of JAK1 and JAK2 in Articular Chondrocytes--
To
investigate the mechanisms of IL-6/sIL-6R effects on chondrocytes,
IL-6/sIL-6R-treated cells were analyzed for activation of JAK1 and JAK2
in a dose- and time-dependent manner. BAC were first
exposed for 10 min to increasing concentrations of IL-6 (100, 200, and
500 ng/ml), in the presence of either 200 or 500 ng/ml sIL-6R, and
protein extracts were analyzed by Western immunoblotting. Activation of
JAK1 and JAK2 reached a maximum for the combination IL-6/sIL-6R at 500 ng/ml of each component, although the phosphorylation was already
detectable for association of lower levels, such as 100 ng/ml IL-6 and
200 ng/ml sIL-6R, particularly for JAK1 (Fig. 2A). The
amounts of respective total proteins remained generally unaffected
(lower panels of each blot). Kinetic studies were then undertaken, using the maximum concentration, i.e. 500 ng/ml
of both IL-6 and sIL-6R, and different time periods (5-60 min). As seen in Fig. 2B, IL-6/sIL-6R-stimulated phosphorylation of
JAK1 occurred within 5 min, peaked at 15 min, and rapidly declined to a
nondetectable level. JAK2 activation was more sustained in BAC, showing
a low level of phosphorylation in controls that suggested the presence
of some amount of constitutively active form. A maximum is reached at 5 min, thus earlier than for JAK1.
IL-6/sIL-6R Induces Activation of the MAP Kinase Pathway
in Articular Chondrocytes--
In addition to activating JAKs,
stimulation of BAC with IL-6/sIL-6R elicited the MAPK pathway (Fig.
3). Indeed, IL-6/sIL-6R treatment caused
specific threonine/tyrosine phosphorylation of ERK1 and ERK2 in a
dose-dependent manner (Fig. 3A, upper
panel), a maximum being reached with addition of 500 ng/ml of both
IL-6 and sIL-6R. This effect was also evidenced by the ability of cell lysates to phosphorylate MBP, a known substrate of in vitro
MAPK assay (Fig. 3A, lower panel). There was no
absolute correlation between Western blotting and MBP assay, regarding
the peak of activity, probably because other MBP kinases beside ERK1
and ERK2 are contributing to MBP phosphorylation (50). The time course of the effects of IL-6/sIL-6R on ERK1/ERK2 phosphorylation and total
MAPK activity is shown in Fig. 3B, upper and lower
panels, respectively. The phosphorylation of ERK1 and ERK2 showed
a plateau of maximum stimulation within 5-15 min, which was maintained
for up to 60 min.
IL-6/sIL-6R Induces STAT1 and STAT3 Activation in
Articular Chondrocytes--
To gain insight into the mechanism of
IL-6/sIL-6R signaling in BAC, cells were treated for different time
periods (5-60 min) with 500 ng/ml of both IL-6 and sIL-6R, and the
STAT1 and STAT3 phosphorylation status in the cytosol and in the
nucleus was analyzed by Western blotting of the related extracts. After
5 min of treatment, STAT1 and STAT3 levels decreased in the cytosol,
whereas their phosphorylation increased in parallel (Fig.
4A, upper panel). This IL-6/sIL-6R-induced tyrosine phosphorylation of STAT1/STAT3 in the
cytosol was accompanied by its translocation to the nucleus (Fig.
4A, lower panel). Comparison between the time
course of the cytosolic and the nuclear extracts suggests that nuclear
translocation reached maximum by ~30 min. It must be noted that
levels of STAT1/STAT3 reincreased in the cytosolic extracts after 60 min, more probably reflecting a redistribution of the proteins to this
cellular compartment. In some of these experiments, a low level of
STAT3 was detected in the nucleus extracts of untreated cultures,
attributable to small basal activation.
To determine whether IL-6/sIL-6R induced the DNA binding activity of
STAT1 and STAT3, we examined the kinetics of STAT1/STAT3 binding
activity in treated BAC. Nuclear extracts were prepared from cells
exposed to IL-6/sIL-6R for different time periods, in the same
conditions as above, and were subjected to EMSA analysis, using
32P-labeled oligonucleotides containing the STAT1 and STAT3
consensus sequences as respective probes. STAT1 binding activity was
significantly increased over the control level, within 5-30 min, which
correspond to the same time period where nuclear translocation was
observed in the Western blots (Fig. 4B, upper
panel). This binding was efficiently competed with a 50-fold molar
excess of the same unlabeled oligonucleotide (Fig. 4B,
upper panel, lane 7), but not with 50-fold excess
of a mutant unlabeled probe (Fig. 4B, upper
panel, lane 8). Similar results were found for STAT3
binding activity (Fig. 4B, lower panel). As shown
in Fig. 4C, supershift analysis confirmed the identity of
the complexes revealed by the previous EMSA study.
IL-6/sIL-6R Decreases Type II Collagen, Aggrecan Core
and Link Protein mRNA Expression in Articular Chondrocytes--
To
determine the effects of IL-6/sIL-6R on type II collagen, aggrecan core
and link protein gene expression, we examined the steady-state levels
of the corresponding mRNAs in BAC, which were serum-deprived and
treated for 24 h with increasing concentrations of IL-6/sIL-6R
(50, 100, 200 ng/ml each). The level of type II collagen, aggrecan core
and link protein mRNAs was determined by Northern blotting and
normalized to the level of GAPDH mRNA. This treatment resulted in a
marked decrease of steady-state levels of these three
cartilage-specific mRNAs (Fig. 5).
Thus, 50/50 ng/ml IL-6/sIL-6R already decreased type II collagen
mRNA levels and the effect was so marked for greater concentrations
(100/100 and 200/200 ng/ml) that the signal was almost undetectable.
Likewise, but to a lower extent, aggrecan core protein mRNA levels
were dose dependently reduced after incubation with 50/50, 100/100, and
200/200 ng/ml IL-6/sIL-6R. Similar results were observed for the link
protein mRNA, albeit with smaller effect.
Parthenolide and PD098059 Inhibit JAK/STAT and MAPK
Pathways, Respectively, in Articular Chondrocytes--
To further
investigate the potential role of IL-6/sIL-6R-induced activation of
JAK/STAT and MAPK pathways in the control of type II collagen and
aggrecan core and link protein gene expression in articular
chondrocytes, we used inhibitors, parthenolide and PD098059, to
selectively block these signaling pathways. Because parthenolide was
shown to also block NF
Next, we wanted to verify that these inhibitors were efficient in the
experimental conditions used here on articular chondrocytes. Thus,
serum-deprived BAC were incubated for 2 h with or without 50 µM parthenolide or 25 µM PD098059, or with
0.5% Me2SO as the control vehicle of these compounds. They
were then subjected to IL-6/sIL-6R (100/100 ng/ml) for 30 min. Effects
on STAT1 and STAT3 DNA binding activity were analyzed by EMSA, whereas
ERK1 and ERK2 activity was studied by Western blotting, together with
determination of total MAPK activity by MBP in vitro assay.
As can be seen on Fig. 7A,
STAT1 and STAT3 DNA binding was completely abrogated by the
parthenolide treatment, whereas it was not affected by PD098059. We
demonstrated likewise that parthenolide blocked STAT1 and STAT3
phosphorylation (data not shown). Additionally, parthenolide was still
efficient in the presence of PD098059, suggesting that this latter does
not interfere with the parthenolide effect on STAT DNA binding.
Although not completely inhibited, ERK activity was markedly reduced at
the PD098059 concentration used here, as revealed by Western blotting
(Fig. 7B). It must be noted that concentrations greater than
25 µM PD098059 were difficult to handle because of low
solubility in aqueous medium. On the other hand, parthenolide by itself
was found to stimulate the basal MAPK activity. The data were also
confirmed by the results obtained with MBP in vitro kinase
assay (Fig. 7B).
Parthenolide, but Not PD098059, Abrogates
IL-6/sIL-6R-induced Down-regulation of Type II Collagen,
Aggrecan Core and Link Protein Transcription in Articular
Chondrocytes--
To further study the mechanism of IL-6/sIL-6R
signaling leading to type II collagen, aggrecan core and link protein
mRNA down-regulation, BAC were exposed for 24 h to IL-6/sIL-6R
at the concentration of 100/100 ng/ml, alone or in combination with the
STAT inhibitor parthenolide (50 µM) or MAPK inhibitor
PD098059 (25 µM), and Northern blotting of RNA was
performed as previously described.
It was found that parthenolide alone was capable of inducing a
significant increase of the basal level of type II collagen mRNA.
Furthermore, it completely abrogated the IL-6/sIL-6R down-regulation of
the collagen message (Fig.
8A). PD098059 alone was
without significant effect on the basal collagen mRNA level,
related to the controls (Me2SO-containing cultures). This
inhibitor was also unable to influence the IL-6/sIL-6R-induced
inhibition. Similar results were obtained for aggrecan core and link
protein mRNA steady-state levels (Fig. 8, A and
B). When both inhibitors were combined, the IL-6/sIL-6R
inhibitory effect was abolished for the three mRNA. These findings
demonstrate that the IL-6/sIL-6R inhibitory effect on type II collagen,
aggrecan core and link protein gene transcription in chondrocytes is
mediated by the JAK/STAT pathway, whereas the MAPK signaling is not
essential for these mechanisms.
Sox9 Expression Is Down-regulated by
IL-6/sIL-6R--
To determine a potential effect of
IL-6/sIL-6R on the expression of Sox9, a key factor that could be
implicated in the observed down-regulation of the cartilage-specific
genes produced by chondrocytes (type II collagen, aggrecan), we
analyzed the steady-state levels of Sox9 mRNA by semiquantitative
reverse transcriptase-PCR and the protein amounts by Western blotting
(Fig. 9, A and B).
We found a parallel decrease of both mRNA and protein levels under the effect of IL-6/sIL-6R. As expected, IL-1 We show here that IL-6 activates JAK/STAT and MAPK pathways in the
primary bovine articular chondrocyte system. Furthermore, blockage of
STAT phosphorylation by parthenolide results in the inhibition of
IL-6-induced down-regulation of type II collagen, aggrecan core and
link protein gene expression. On the other hand, inhibition of MAP
kinase pathway by PD 098059 does not significantly alter the cytokine
effect on transcriptional activity of these matricial genes. These
results demonstrate for the first time that the JAK/STAT pathway is
essential for IL-6 signaling responses in chondrocytes, including
down-regulation of cartilage-specific ECM components.
Chondrocytes play an essential role in regulating the balance between
ECM degradation and synthesis and their primary function to maintain
cartilage ECM integrity is significantly altered in both inflammatory
and degenerative joint diseases because of changed levels of local
growth factors and cytokines (53, 54). IL-6 was initially considered as
a proinflammatory cytokine-like tumor necrosis factor- IL-6, as other members of the IL-6-type cytokines, including oncostatin
M, are known to induce the JAK/STAT signaling cascade in other systems
(2) but this has not been shown in chondrocytes. Our demonstration that
IL-6 triggers phosphorylation of JAK1, JAK2, STAT1, and STAT3 in
primary cultures of articular chondrocytes suggest that these cells,
together with the synoviocytes, could be a source of activated STAT1
and STAT3 found in the synovial fluid and fibroblasts of patients with
RA (58, 59). Interestingly enough, we demonstrate here for the first
time that the IL-6-induced activation of the JAK/STAT pathway results
in down-regulation of three important specific genes of cartilage, type
II collagen, aggrecan core and link proteins, suggesting that IL-6 not
only contributes to cartilage erosion by inducing degradative enzymes but also via reduction of ECM synthesis. Indeed, parthenolide, a
sesquiterpene lactone previously found as an effective inhibitor of
IL-6-type cytokine by blocking STAT phosphorylation (43), was capable
of abolishing the STAT DNA-binding and the subsequent IL-6 inhibition
of the three ECM genes transcription. Although the inhibitor was also
shown to block the activation of NF- We found here that in the presence of parthenolide, the MAP kinase
pathway was already activated in the controls (in the absence of
IL-6/sIL-6R), as shown by increased phosphorylation of ERK1 and ERK2 on
the Western blots. This clearly indicates that an interaction exits
between the JAK/STAT and MAPK pathways in articular chondrocytes. To
this regard, it has been demonstrated in a recent report that there is
a balanced activation of the SHP-2/Ras/Erk and STAT signaling
cascades emanating from gp130 (63). The authors have generated mice
with a COOH-terminal gp130 Our study indicates that Sox9, a key factor of type II collagen and
aggrecan expression, is down-regulated by IL-6/sIL-6R association at
both mRNA and protein levels. This suggests that Sox9 may play a
role in the molecular mechanism whereby the cartilage-specific matrix
gene expression is inhibited by IL-6 associated to its soluble
receptor, through the JAK/STAT signaling pathway. The observed
correlation between Sox9 inhibition and down-regulation of matrix gene
expression under the IL-6/sIL-6R effect suggests a link between this
transcription factor and the STAT proteins in the mechanisms
controlling the transcriptional activity of these matricial genes. How
Sox9 may cooperate with the STATs at the level of DNA-binding activity
and subsequent modulation of type II collagen, aggrecan core and link
protein gene transcription remains to be elucidated.
Finally, our data strongly suggest that the activation of the JAK/STAT
pathway by inflammatory cytokines of the IL-6-type mediates a change in
the chondrocyte phenotype and loss of cartilage matrix by inhibiting
the expression of specific cartilage matrix molecules, including type
II collagen, aggrecan core and link proteins, that maintain the
differentiated phenotype of chondrocytes. Therefore, inhibiting this
signaling pathway may be an effective approach in preventing cartilage
alteration induced by IL-6-type cytokines in arthritides, as it has
been recently proposed (65-69). However, because of the cross-talk
between ERK and STAT pathways, already mentioned above, a combination
of selective blockers may be necessary.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and anti-IL-1 antibodies has been shown
to be effective on this established murine model (26, 27), IL-6 may
even act earlier in the course of the disease than those of tumor
necrosis factor-
and IL-1 (22). First treatment of human RA patients
with mouse monoclonal anti-IL-6 antibody or with humanized anti-IL-6R
antibody have resulted in improvement of symptoms and laboratory
findings (28, 29).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was a generous gift from Dr. Soichiro Sato (Shizuoka, Japan). Mouse monoclonal
anti-phosphotyrosine antibody (4G10), mouse monoclonal anti-phospho-MAP
kinase (specific for threonine and tyrosine-phosphorylated residues of
ERK1/ERK2), rabbit anti-MAP kinase 1/2 (ERK1/2-CT), and rabbit
polyclonal anti-human TYK2 antibodies were purchased from Upstate
Biotechnology, Inc. Rabbit polyclonal anti-JAK1 (HR-785) and anti-JAK2
(C-20), mouse monoclonal anti-STAT1 p84/p91 (C-136), anti-STAT3 (F-2
or C-20), anti-phosphospecific STAT1-Tyr701 (A-2),
STAT3-Tyr705 (B-7), anti-
-tubulin (H-235), and nonimmune
serum were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg,
Germany). Rabbit polyclonal anti-I
B
was obtained from Cell
Signaling Technology. Rabbit polyclonal anti-Sox9 antibody was kindly
provided by B. de Crombrugghe (Department of Molecular Genetics,
University of Texas M.D. Anderson Cancer Center, Houston, TX). Primary
antibodies were revealed with anti-rabbit or anti-mouse horseradish
peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology,
Inc.), using an ECL+Plus Western blot detection kit (Amersham
Biosciences, Orsay, France). The oligonucleotide probes: STAT1
(5'-CATGTTATGCATATTCCTGTAAGTG-3'), mutant STAT1
(5'-CATGTTATGCATATTGGAGTAAGTG-3'), STAT3 (5'-GATCCTTCTGGGAATTCCTA-3'), mutant STAT3 (5'-GATCCTTCTGGGCCGTCCTA-3'), and NF
B
(5'-AGTTGAGGGGACTTTCCCAGGC-3') were supplied by Invitrogen
(Cergy Pontoise, France). All other chemicals were of the highest
purity available and were from Sigma.
-32P]ATP (PerkinElmer Life
Sciences). After 30 min at 30 °C, the reaction was stopped with 5 µl of Laemmli buffer and the sample was submitted to SDS-PAGE. The
gel was then dried and subjected to autoradiography. The intensity of
32P-labeled phosphorylated MBP bands was measured by
scanning x-ray films (Kodak, X-Omat) and quantified by the ImageQuant
program (Amersham Biosciences).
-32P]ATP (25 fmol) using T4 polynucleotide kinase
(Invitrogen). Final binding reactions were performed in 13 mM Hepes (pH 7.9), 65 mM NaCl, 0.15 mM EDTA, 8% glycerol, 0.02% Nonidet P-40, 1 mM DTT, and 0.05 µg/µl poly(dI-dC) for STAT1 and STAT3,
and in 20 mM Hepes (pH 7.5), 50 mM KCl, 4 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.05% Nonidet P-40, 20% glycerol, 1 mg/ml bovine
serum albumin, 0.025 mM poly(dI-dC) for NF
B. Supershift
experiments were performed by incubating the nuclear extracts with
nonimmune IgG, anti-STAT1 antibody, or anti-STAT3 antibody for 2 h
at room temperature before addition of the labeled probe. The samples
were then submitted to a 6 or 8% PAGE in 0.5× TBE (45 mM
Tris (pH 7.8), 45 mM boric acid, and 1 mM EDTA)
and visualized by autoradiography (46).
-actin, sense
5'-GTGGGGCGCCCCAGGCACCA-3', antisense 5'-CTCCTTAATGTCACG CACGATTTC-3'. The following amplification protocol was used: 1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C. PCR products
were analyzed in 2% agarose gels after staining with ethidium bromide. Densities of the band were quantified using ImageQuant software (Amersham Biosciences) and the relative expression of messengers studied was calculated as relative ratio to
-actin. The procedure was repeated on three different experiments with always the same findings.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of IL-6 and/or sIL-6R on tyrosine
phosphorylation of JAKs. A and B, after a
16-h preincubation in serum-free medium, chondrocytes were stimulated
with or without IL-6 (500 ng/ml)/sIL-6R (500 ng/ml) for 10 min. Then,
they were lysed, and the extracts were immunoprecipitated (IP) with
anti-JAK1 or anti-JAK2 antibodies and immunoblotted (blot) with
anti-Tyr(P) antibody, as described under "Experimental Procedures."
The same membranes were then reblotted with anti-JAK1 or -JAK2
antibodies, respectively. C, after a 16-h preincubation in
serum-free medium, chondrocyte (lane 1) or control HepG2
cell (lane 2) extracts were lysed, immunoprecipitated
(IP) with anti-TYK2 antibody, and immunoblotted
(blot) with the same antibody, as described under
"Experimental Procedures."
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Fig. 2.
Effect of IL-6/sIL-6R on tyrosine
phosphorylation of JAK1 and JAK2. A, dose-response of JAK1
and JAK2 tyrosine phosphorylation to IL-6/sIL-6R treatment.
Serum-deprived chondrocytes were stimulated for 10 min with increasing
concentrations of IL-6 (0, 100, 200, or 500 ng/ml) and sIL-6R (0, 200, or 500 ng/ml). Cell extracts were prepared and immunoprecipitated
(IP) with anti-JAK1 or -JAK2 antibodies. Immunoprecipitated
proteins were analyzed by SDS-PAGE, followed by Western blotting
(blot), using antibody to phosphotyrosine. Then, the
membranes were stripped and probed again with antibodies to JAK1 or
JAK2, respectively. B, time course of JAK1 and JAK2 tyrosine
phosphorylation by IL-6/sIL-6R. Serum-deprived chondrocytes were
stimulated with IL-6 (500 ng/ml)/sIL-6R (500 ng/ml) for 5, 15, 30, and
60 min. Immunoprecipitation and Western blotting were performed as
described above. See "Experimental Procedures" for details.
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Fig. 3.
Effect of IL-6/sIL-6R on MAP kinase
activation. A, dose response of MAP kinase activation
to IL-6/sIL-6R treatment. Serum-deprived chondrocytes were stimulated
for 10 min with increasing concentrations of IL-6 (0, 100, 200, or 500 ng/ml) and sIL-6R (0, 200, or 500 ng/ml). For Western blot (upper
panel), cytoplasmic extracts, obtained with hypotonic buffer, were
subjected to SDS-PAGE. They were successively immunoblotted with
anti-phospho-ERK1/ERK2 (blot:
-pERK1/2) and anti-ERK1/ERK2 (blot:
-ERK1/2) antibodies to verify equal loading.
For in vitro kinase assay (lower panel), cellular
extracts obtained with RIPA buffer were subjected to MAP kinase assay
using MBP as a substrate. The histograms represent the levels of
phosphorylated MBP, obtained by quantifying the intensity of the
MBP-phosphorylated bands (indicated by an arrow) in the
autoradiography. B, time course of MAP kinase activation by
IL-6/sIL-6R. Cultures of chondrocytes were exposed to IL-6 (500 ng/ml)/sIL-6R (500 ng/ml) for 5, 15, 30, and 60 min in the same
conditions as previously. Western blot (upper panel) and
in vitro MAP kinase assay (lower panel) were
performed as described above. See "Experimental Procedures" for
details.
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Fig. 4.
Effect of IL-6/sIL-6R on STAT
activation. Serum-deprived chondrocytes were stimulated with IL-6
(500 ng/ml)/sIL-6R (500 ng/ml) for 5, 15, 30, and 60 min. Cytoplasmic
and nuclear extracts were obtained with hypotonic and hypertonic buffer
as described under "Experimental Procedures." A, STAT1
and STAT3 tyrosine phosphorylation and translocation from cytosol to
the nucleus in chondrocytes treated with IL-6/sIL-6R. Cytoplasmic
(upper panel) and nuclear (lower panel) fractions
were resolved by SDS-PAGE and successively immunoblotted
with anti-phospho-STAT1 (blot: -pTyrSTAT1),
anti-STAT1 (blot:
-STAT1), anti-phosphoSTAT3
(blot:
-pTyrSTAT3), and anti-STAT3
(blot:
-STAT3). B, STAT1 and STAT3
binding activity in chondrocytes treated with IL-6/sIL-6R. Nuclear
extracts of control (
) (lanes 2 and 10) and
stimulated chondrocytes (lanes 3-8 and 11-16)
were subjected to EMSA using
-32P-labeled DNA probes of
STAT1 (upper panel) and STAT3 (lower panel).
Fifty-fold excess of corresponding unlabeled probes (lanes 7 and 15) or mutant unlabeled probes (lanes 8 and
16) were used for competitive inhibition assay. Lanes
1 and 9 were loaded with only radiolabeled probes.
Arrows indicate complexes of nuclear extracts with STAT1 or
STAT3. C, nuclear extracts of stimulated cells (30 min) were
preincubated with a nonimmune serum, with anti-STAT1 or anti-STAT3
antibodies for 2 h and subjected to EMSA using
-32P-labeled DNA probes of STAT1 (upper
panel) and STAT3 (lower panel). Arrows
indicate complexes of nuclear extracts with STAT1 or STAT3. See
"Experimental Procedures" for details.
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Fig. 5.
Effect of IL-6/sIL-6R on type II collagen,
aggrecan and link protein mRNA expression. Serum-deprived
chondrocytes were stimulated for 24 h with or without IL-6 (50, 100, and 200 ng/ml)/sIL-6R (50, 100, and 200 ng/ml). Total RNA was
isolated from the chondrocyte cultures and analyzed by Northern
blotting. The blot was hybridized with specific
-32P-labeled probes for type II collagen (coll2),
aggrecan, and GAPDH mRNA, which served as a control for sample
loading (A), and for link protein and GAPDH mRNA
successively (B). See "Experimental Procedures" for
details.
B activation (51), we wanted to determine
whether IL-6/sIL-6R could activate NF
B in our experimental
conditions (Fig. 6). Effects of
IL-6/sIL-6R (100/100 ng/ml) on NF
B activity were analyzed by EMSA
and by I
B Western blotting after 30 or 60 min exposure. As shown in
Fig. 6, both EMSA and Western blot analyses did not reveal any
significant change between controls and IL-6/sIL-6R-treated
chondrocytes, attesting that IL-6/sIL-6R did not activate NF
B in the
present conditions, whereas IL-1
, which is well known for its
ability to stimulate NF
B, was found efficient in the same conditions (positive control).
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Fig. 6.
Effect of IL-6/sIL-6R on
NF B activation. Serum-deprived
chondrocytes were stimulated for 30 and 60 min with IL-6 (100 ng/ml)/sIL-6R (100 ng/ml) or with IL-1
(10 ng/ml) as a positive
control. Cytoplasmic and nuclear extracts were obtained with hypotonic
and hypertonic buffer as described under "Experimental Procedures."
A, effect on NF
B DNA binding. Nuclear extracts of control
(0) and stimulated chondrocytes (30 min) were subjected to EMSA using
-32P-labeled DNA probes of NF
B. p65 and p50 indicate
the subunits of the NF
B system. B, effect on I
B
degradation. Cytoplasmic extracts were subjected to SDS-PAGE and
immunoblotted with anti-I
B
antibody.
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Fig. 7.
Effect of parthenolide and PD098059 on
JAK/STAT and MAP kinase pathways. Serum-deprived chondrocytes were
incubated for 2 h with or without Me2SO (0.5% (v/v)
final concentration), parthenolide (Pa, 50 µM)
and PD098059 (PD, 25 µM). Then, they were
stimulated (+) or not ( ) with IL-6 (100 ng/ml)/sIL-6R (100 ng/ml) for
30 min. Cytoplasmic and nuclear extracts were obtained with hypotonic
and hypertonic buffers as described under "Experimental
Procedures." A, effects of parthenolide and PD098059 on
STAT1 and STAT3 binding activity. Nuclear extracts of control (
)
(lanes 1, 3, 5, 7,
9, 12, 14, 16, 18, and
20) and stimulated chondrocytes (+) (lanes 2,
4, 6, 8, 10,
13, 15, 17, 19, and
21) were subjected to EMSA using
-32P-labeled
DNA probes of STAT1 (left) and STAT3 (right).
Lanes 11 and 22 were loaded with only
radiolabeled probes. Arrows indicate complexes of nuclear
extracts with STAT1 or STAT3. B, effects of parthenolide and
PD098059 on ERK1/ERK2 phosphorylation and MAP kinase activity.
Cytoplasmic extracts were subjected to SDS-PAGE. They were successively
immunoblotted with anti-phospho-ERK1/ERK2 (blot:
-pERK1/2) and anti-ERK1/ERK2 (blot:
-ERK1/2) antibodies to verify equal loading. For in
vitro kinase assay (lower panel), cellular extracts
were subjected to MAP kinase assay using MBP as a substrate. The
histograms represent the levels of phosphorylated MBP, obtained by
quantifying the intensity of the MBP-phosphorylated bands (indicated by
an arrow) in the autoradiography. See "Experimental
Procedures" for details.
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Fig. 8.
Effect of parthenolide and PD098059 on
IL-6/sIL-6R induced down-regulation of type II collagen, aggrecan and
link protein mRNA expression. Serum-deprived chondrocytes were
incubated for 2 h with or without Me2SO (0.5% (v/v)
final concentration), parthenolide (Pa, 50 µM)
and PD098059 (PD, 25 µM). Then, they were
stimulated (+) or not ( ) with IL-6 (100 ng/ml)/sIL-6R (100 ng/ml) for
24 h. Total RNA was isolated from the chondrocyte cultures and
analyzed by Northern blotting. The blot was hybridized with specific
-32P-labeled probes for type II collagen (coll2),
aggrecan, and GAPDH mRNA (A), and for link protein and
GAPDH mRNA successively (B). See "Experimental
Procedures" for details.
also exerted an inhibition on Sox9 expression in the same experimental conditions (52).
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Fig. 9.
Effect of IL-6/sIL-6R on Sox9
expression. Serum-deprived chondrocytes were stimulated with IL-6
(100 ng/ml)/sIL-6R (100 ng/ml) or with IL-1 (10 ng/ml) for 24 h. A, cellular extracts (30 µg of protein obtained with
RIPA buffer) were subjected to SDS-PAGE and immunoblotted with
anti-Sox9 and anti-
-tubulin as a control for protein loading.
B, total RNA was used for reverse transcriptase
(RT)-PCR assay of SOX9 and
-actin mRNA steady-state
levels. PCR reactions were carried out at increasing numbers of cycles
(20, 23, 26, 29, and 32 cycles for
-actin and 25, 28, 31, 34, and 37 cycles for SOX9) to check the linearity of amplification before
analysis of transcript scanning. Results are expressed as a
representative histogram of Sox9 relative expression versus
-actin.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and IL-1,
because of its IL-1-like effects on immune and hepatic cells. Recently,
the cytokine has been shown to enhance the aggrecanase-mediated
proteoglycan catabolism in articular cartilage (55). Elevated IL-6
levels in serum and synovial fluids correlate with inflammatory and
erosive arthritides (11, 16-20). Furthermore, IL-1 and tumor necrosis
factor-
can also stimulate the production of IL-6 by articular
cartilage chondrocytes (56, 57). As previously reported by others (34,
55), we found that the effect of IL-6 requires the presence of its
soluble receptor (sIL-6R) in the culture medium. This is probably
because of the fact that levels of membrane-anchored IL-6 receptor
(gp80) on chondrocytes are very low compared with those on other cell types such as hepatocytes (34). However, high amounts of sIL-6R are
found in synovial fluid of arthritic patients (5-40 ng/ml) (11) and
are likely to participate in the signaling of IL-6 in both synovial
cells and articular chondrocytes. Although the concentrations used
here, which were chosen to demonstrate more efficiently the IL-6
action, may appear greater than those found in the joint of arthritic
patients, it was clear here that the effect could already be detected
for equivalent doses.
B in other systems, we did not
detect any significant effect of IL-6 on this transcription factor in
our experimental conditions. Interestingly enough, putative STAT3
regulatory sites have been already reported in the promoter or first
intron regions of the genes for rat type II collagen, human and rat
link protein, and rat stromelysin (metalloprotease-3), suggesting that
these are target genes for the JAK-STAT signaling pathway (60, 61). Similarly, a potential STAT sequence has been found in the promoter of
the human aggrecan gene, which may confer responsiveness to cytokines
of the IL-6 family (62). Work is in progress to determine whether these
putative STAT binding sites are actually implicated in the expression
control of these genes in articular chondrocytes. However, the fact
that the promoters of these genes share similar STAT elements strongly
suggests that they are coordinately regulated by IL-6, as observed in
this study.
STAT "knock-in" mutation
that deletes all STAT-binding sites, but still permits activation of
SHP-2/Ras/Erk pathway. Gp130
STAT mice phenocopied mice
deficient for IL-6 (impaired humoral and mucosal immune and hepatic
acute phase responses) and LIF (failure of blastocyst implantation).
However, unlike mice with null mutations in any of the components of
the gp130 signaling pathway, gp130
STAT mice also
displayed gastrointestinal ulceration and a severe joint disease with
features of chronic synovitis and degradation of the articular
cartilage. Interestingly enough, a mitogenic hyper-responsiveness of
synovial cells was found, resulting from the sustained gp130-mediated
SHP-2/Ras/Erk activation because of impaired STAT-mediated induction of
suppressor of cytokine signaling proteins, which normally limits gp130
signaling. These data identified the importance of STAT signals in
promoting inhibitory feedback signals on gp130 and the associated
SHP-2/Ras/Erk pathway, thereby reinforcing the concept that the two
pathways are under reciprocal negative control (64).
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ACKNOWLEDGEMENTS |
---|
We acknowledge the helpful suggestions of Dr. Sandra Pellegrini (Pasteur Institute, Paris) and Dr. Patrick Mayeux (Institute of Molecular Biology, Cochin Hospital, Paris). We also thank Pr. B. de Crombrugghe for providing the Sox9 antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by the Lower Normandy Regional Council.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 Lower Normandy Regional Council.
To whom correspondence and reprint requests should be
addressed: Laboratoire de Biochimie du Tissu Conjonctif, Faculté
de Médecine, Niveau 3, 14032 Caen Cedex, France. Tel.:
33-02-31-06-82-18; Fax: 33-02-31-06-82-24; E-mail:
pbogdanowicz@hotmail.com.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M110773200
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ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
BAC, bovine articular chondrocytes;
Me2SO, dimethyl
sulfoxide;
ECM, extracellular matrix;
ERK, extracellular
signal-regulated kinase;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
IB, inhibitor of NF
B;
JAK, Janus kinase;
MAPK, mitogen-activated protein kinase;
MBP, myelin basic protein;
MOPS, 3-(N-morpholino)propanesulfonic acid;
NF
B, nuclear factor
for
light chain in B cells;
PD 098059, Park Davis 098059;
sIL-6R, soluble interleukin-6 receptor;
Sox9, Sry-type HMG box-9;
STAT, signal
transducer and activator of transcription;
TYK, tyrosine kinase;
gp, glycoprotein;
EMSA, electrophoretic mobility shift assay;
DTT, dithiothreitol;
RA, rheumatoid arthritis.
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