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Departments of
*
Medicine and
Surgery, Research Center and Notre-Dame Hospital of the Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(6, 7, 8), and MMPs are found elevated in patients with
arthritis (8, 9, 10), they may contribute directly to
progression of the disease. Identification of MMP-inducing agents and
deciphering the mechanisms of induction are necessary for developing
novel strategies to block cartilage resorption. The TIMP multigene family consists of TIMP-1, -2, -3, and -4, which by complexation with MMPs regulate enzyme-inhibitor balance (3, 11). MMP inhibitors are potentially important therapeutic agents for arthritis (12). TIMPs also inhibit tumor growth, invasion, and metastasis and have growth-promoting and antiangiogenic activities (11, 13). TIMP-3 was originally isolated from chicken fibroblasts and is distinctly associated with ECM (14, 15). It is expressed in several normal and tumor tissues (16, 17, 18). By its strategic location in ECM, TIMP-3 blocks tumor growth and invasion possibly by inhibiting angiogenesis (19) and by inducing apoptosis (20). TIMP-3 inhibits MMPs similarly to TIMP-1 (21) and is extensively expressed in joint tissues (22). Due to its potential for inhibiting cartilage resorption, elucidation of signaling mechanisms regulating TIMP-3 by arthritis-associated stimuli is of particular interest.
Oncostatin M (OSM) produced by activated monocytes and T lymphocytes (23) is a multifunctional cytokine that affects the growth and differentiation of several cell types (24, 25). OSM belongs to the IL-6 family of cytokines, which includes IL-11 and LIF. They have a similar helical structure and share the receptor gp130 (25). An anti-inflammatory role for OSM in vivo was suggested because it suppressed inflammation and tissue damage in a mouse model of RA (26). OSM was undetectable in the synovial fluid of osteoarthritis but was present in the fluid of RA patients (27). Its mRNA and protein were elevated in the RA synovial cells (28, 29). Injection of human OSM into goat joints promoted cartilage resorption and inhibited synthesis of proteoglycans (30), suggesting its implication in the pathogenesis of rheumatoid joint lesion. OSM induced TIMP-1 in human chondrocytes (31), TIMP-1 and MMP-13 in rat osteoblasts (32), and MMP-1 in human synovial fibroblasts (33). The mechanisms by which OSM regulates TIMPs and MMPs are poorly understood. We recently showed the involvement of tyrosine kinases in OSM induction of TIMP-3 in articular chondrocytes (34); however, precise signaling pathways have not been identified. Janus kinase (JAK)/STAT signaling pathway is activated by IFNs and IL-6-type cytokines (35, 36, 37). The ligand-receptor interaction brings assembly of cytokine receptors, receptor-associated JAKs that recruit and activate STAT proteins. Phosphorylated STATs then dimerize, translocate to the nucleus, and direct transcription of the specific target genes (36, 37). In the present study, we used two important chondrocyte model systems, namely, bovine and human primary articular chondrocyte, and showed induction of multiple signaling cascades and a crucial role of the JAK/STAT pathway in the OSM induction of TIMP-3 and MMP gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture supplies such as DMEM, FCS, Fungizone, and
penicillin-streptomycin were purchased from Life Technologies
(Burlington, Ontario, Canada). Collagenase type II and curcumin were
obtained from Sigma (St. Louis, MO). JAK3 inhibitor,
4-(4'-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline (38)
and AG490, -cyano-(3,4-dihydroxy) N-benzylcinnamide
(39), were obtained from Calbiochem (San Diego, CA). Human
recombinant OSM was purchased from R&D Systems (Minneapolis, MN). The
anti-human STAT1, extracellular signal-regulated kinase (ERK) 1/2,
dual-phosphospecific ERK1/2
(Thr202/Tyr204), p38
(Thr180/Tyr182), c-Jun
N-terminal kinase (JNK;
Thr183/Tyr185), and
phosphospecific STAT1 (Tyr701) rabbit polyclonal
Abs were provided by New England Biolabs (Mississauga, Ontario,
Canada). The anti-mouse JAK1, dual-phosphospecific JAK1
(Tyr1022/Tyr1023), JAK2
(Tyr1007/Tyr1008), and
anti-human JAK3 rabbit polyclonal Ab was purchased from Quality
Controlled Biochemicals (Hopkinton, MA). Anti-mouse JAK2 rabbit
polyclonal and anti-phosphotyrosine mAb 4G10 were obtained from
Upstate Biotechnology (Waltham MA). Agarosre-conjugated rabbit
anti-human JAK3 and STAT1-binding consensus oligonucleotide were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein
A-Sepharose beads were obtained from Pharmacia-Amersham Biotech
(Piscataway, NJ). HRP-conjugated sheep anti-rabbit IgG and a
chemiluminescence detection system was purchased from Roche Molecular
Biochemicals (Laval, Quebec, Canada). The gel shift assay core system
was purchased from Promega (Madison, WI).
[-32P]CTP was obtained from DuPont-NEN
(Boston, MA).
Primary cultures of chondrocytes
Normal bovine articular cartilage was obtained from the knee and hip joints of freshly slaughtered adult animals through a local slaughterhouse. Human cartilage was from the femoral heads of patients who underwent hip replacement surgery as a result of osteoarthritis or RA at the Notre-Dame Hospital. The cartilage with bones were dipped in 1% proviodine for sterilization and washed extensively with water. The slices of cartilage were dissected out, kept for 1 h at 4°C in PBS containing 5x penicillin-streptomycin and 5x Fungizone, and washed five times with large volumes of PBS. Chondrocytes were released from bovine cartilage by digestion with Pronase (1 mg/ml) for 1 h and collagenase type II (2 mg/ml) for 12 h in DMEM at 37°C. The cells were centrifuged and washed five times with PBS and plated at high density. The cells were first allowed to adhere to the plates in DMEM alone for 4 h and then supplemented with 10% serum for confluent growth (up to 6 days). These cells in primary and first-passage cultures maintain their phenotype by expressing chondrocyte-specific type II collagen. Before different treatments, cells were made quiescent in serum-free DMEM for 24 h. The potential inhibitors were added 30 min before the OSM.
RNA extraction and Northern blot analysis
Total RNA from primary cultures of chondrocytes was extracted
using the acid guanidinium method (40), and 5-µg
aliquots were analyzed by electrophoretic fractionation in 1.2%
formaldehyde-agarose gels and transferred to Zeta-probe membranes
(Bio-Rad, Richmond, CA). Northern blot analysis of RNA was performed
with bovine TIMP-3 and human MMP-1, -3, and -13 cDNA probes. The bovine
TIMP-3 plasmid and human MMP-3 plasmid were linearized with
NarI, human MMP-13 plasmid with EcoRI, and RNA
probes were synthesized with T7 RNA polymerase according to the
protocols of Promega Biotech. The human MMP-1 (clone pSP64) was
digested with PstI and a probe was synthesized using T3 RNA
polymerase. The human 28S ribosomal RNA plasmid (American Type Culture
Collection, Manassas, VA) was cut by XbaI and the RNA probe
was synthesized with T7 RNA polymerase. All probes were labeled to
high-specific activity (1 x 108 cpm/µg)
with [-32P]CTP (3000 Ci/mmol) with the RNA
labeling kit from Promega Biotech according to their protocols.
Western blot analysis and immunoprecipitations
After treatment with inhibitors and OSM for various time periods, total cellular protein extracts were prepared from bovine and human chondrocytes as described before (34). Cells were lysed in lysis buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 50 mM DTT, and 1% Triton X-100). Cell lysates were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes by electroblotting, and reacted with phosphorylation-state-specific Abs at concentrations suggested by the manufacturers. Proteins were visualized by HRP-conjugated sheep anti-rabbit IgG and a chemiluminescence detection system. The membranes were stripped with buffer containing 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 6.8) at 55°C for 40 min and reprobed with total Abs.
For immunoprecipitations, chondrocytes were exposed to OSM alone or with curcumin, rinsed twice with ice-cold PBS, and then lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin). Cell lysates were precleared with protein A-Sepharose beads for 30 min at 4°C. After removal of protein A-Sepharose, cell supernatants (400 µg of total proteins) were incubated with agarose-conjugated rabbit anti-human JAK3 Ab for 3 h at 4°C. The immune complexes were washed four times with cold RIPA buffer and the immunoprecipitated proteins were eluted into SDS-PAGE sample buffer (2% SDS, 62.5 mM Tris-HCl (pH 6.8), 50 mM DTT, and 10% glycerol) by heating at 100°C for 5 min, fractionated by 8% SDS-PAGE, and probed with anti-phosphotyrosine Ab (4G10) by immunoblotting. After stripping, these membranes were reprobed for total JAK3.
EMSA
Nuclear extracts were prepared according to details in Ref. 41 , with some modifications. After exposure to inhibitors and OSM, chondrocytes were washed with cold PBS and scraped with 1.5 ml of cold PBS. Cells were centrifuged for 10 s and resuspended in 400 µl of cold buffer A (10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.1% Nonidet P-40, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. Samples were centrifuged for 10 s and the supernatant was discarded. The nuclear pellets were resuspended in 50 µl of cold buffer B (20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and kept on ice for 30 min for high salt extraction. Cellular debris was removed by centrifugation for 20 min and supernatant fraction (nuclear extract) was stored at -70°C. EMSA were performed using a gel shift assay core system as described in Promega protocols. Two micrograms of nuclear proteins was mixed with 32P-end-labeled dsSTAT1 (p84/p91)-binding consensus (underlined) oligonucleotide, 5'-CATGTTATGCATATTCCTGTAAGTG-3', at room temperature for 20 min. The DNA-protein complex formed was separated from free oligonucleotides on 4.5% native polyacrylamide gel. A mutated STAT1-binding consensus oligonucleotide, 5'-CATGTTATGCATATTGGAGTAAGTG-3', was used to examine the specificity of binding of STAT1 to the DNA. The specificity of binding was also examined by competition with the excessive unlabeled oligonucleotide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether chondrocytes from normal bovine and human
arthritic femoral head cartilage respond to OSM, primary cultures of
bovine chondrocytes kept in serum-deprived medium for 24 h were
subjected to 18-h treatment with OSM at different concentrations
(0.1–50 ng/ml). In agreement with our earlier results
(34), Northern blot hybridization analysis of RNA with the
bovine TIMP-3 and human MMP-1, -3, and -13 probes, respectively,
revealed a dose-dependent (at 10–50 ng/ml) induction of TIMP-3, MMP-1,
-3, and -13 mRNAs by OSM (Fig. 1
A). Analysis of chondrocytes
from different patients revealed that MMP-1, -3, and -13 messages were
also induced by the effective dose of 10 ng/ml OSM in human cells (Fig. 1B, also see Figs. 5B and 8B). The
levels of 28S rRNA levels were even. This dose was used in all of the
subsequent parts of the study.
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To investigate the mechanisms of OSM signaling in chondrocytes,
OSM-treated cells were analyzed for activation of JAK1, JAK2 JAK3, and
STAT1. Quiescent bovine and human chondrocytes were exposed to 10 ng/ml
OSM for different time periods (5–60 min), and protein extracts were
analyzed by Western immunoblotting with phosphospecific Abs or
immunoprecipitation with JAK-3 Ab followed by probing with
anti-phosphotyrosine Ab. OSM-stimulated phosphorylation of JAK1,
JAK2, JAK3, and STAT1 occurred within 5 min, peaked between 15 and 30
min, and progressively declined to different extents in bovine and
human chondrocytes. JAK2 activation was more sustained in bovine
chondrocytes, whereas bovine JAK3 could not be analyzed due to its
nonreactivity by immunoprecipitation (Fig. 2). Similar analysis of the three
subclasses of mitogen-activated protein kinases (MAPKs) revealed
activation of ERKs, p38, and JNKs between 5 and 30 min that declined by
60 min (Fig. 3). The levels of respective
total proteins remained generally unaffected (lower
panels of each blot).
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A pharmacologic JAK3 inhibitor specifically blocks its activation
(38) while AG490 is a JAK2-specific inhibitor
(39). To gain insight into the mechanism of OSM signaling
leading to TIMP-3 and MMP RNA up-regulation, cells were exposed to OSM
alone or in combination with the specific JAK3 inhibitor or JAK2
inhibitor AG490. Total cellular protein extracts prepared after 15 min
of OSM treatment were analyzed for STAT1 and ERK activation. As shown
by Western blotting, JAK3 inhibitor dose-dependently reduced
OSM-induced phosphorylation of STAT1, but had no effect on ERK
activation (Fig. 4). AG490 had no impact
on OSM-induced activation of STAT1 and ERKs (Fig. 4). The levels of
STAT1 and ERKs detected by total Abs remained constant and were not
affected by the treatments of chondrocytes from both species. For
TIMP-3 and MMP RNA analysis, total RNA was extracted from chondrocytes
after treatment with OSM or OSM and the inhibitors for 20 h and
probed by Northern blot hybridization. As shown in Fig. 5
, JAK3 inhibitor dose-dependently
suppressed the OSM-induced MMP-1, -3, and -13 gene expression and AG490
had no effect. Basal levels of TIMP-3 message were elevated;
nevertheless, its induction by OSM was similarly inhibited by the JAK3
inhibitor. The 28S rRNA levels were unaffected. These results are in
conformity with those observed for STAT1 activation in Fig. 4 and
suggest JAK3 as a key signaling mediator for TIMP-3/MMP induction by
OSM. As no JAK1-specific inhibitor is currently available, its role
could not be investigated.
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To examine the effect of an anti-inflammatory agent, curcumin
(see Discussion), on OSM-stimulated JAK/STAT and MAPK
pathways, protein extracts from OSM- and curcumin-treated human and
bovine chondrocytes were analyzed for activation of JAK1, JAK2, JAK3,
and STAT1 as well as ERK, p38, and JNK MAPKs. Different concentrations
of curcumin had no effect on the OSM-induced phosphorylation of ERKs,
p38, JAK1, JAK2, and JAK3 (Fig. 6);
however, phosphorylation of JNKs and STAT1 was suppressed in a
dose-dependent fashion. The 15 µM dose of curcumin effectively
inhibited most of the response to OSM. The levels of respective total
proteins were even. Thus, curcumin mimics the effects of JAK3 inhibitor
and is a potent novel inhibitor of the JAK/STAT signaling pathway.
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To determine if by inhibiting OSM-stimulated STAT1 phosphorylation
the JAK3 inhibitor and curcumin affected the DNA-binding capacity of
STAT1, chondrocytes were preincubated for 30 min with curcumin followed
by treatment with OSM for 25 min. Nuclear proteins extracts were then
examined by EMSA for the STAT1 DNA-binding activity. Curcumin, by
itself, had no effect on DNA-binding capacity of STAT1, but at
concentrations of 5–15 µM, dose-dependently down-regulated
OSM-induced DNA binding of STAT1. At a 15 µM concentration, curcumin
completely abolished OSM-activated STAT1 binding to DNA both in bovine
and human cells (Fig. 7). OSM-stimulated
binding of STAT1 to its target DNA was also blocked by the specific
JAK3 inhibitor but not by the JAK2 inhibitor AG490 (Fig. 7A), suggesting that JAK3 may be mediating STAT1
activation.
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To evaluate whether proximal inhibition of STAT1 phosphorylation
and DNA binding by curcumin affects OSM-induced TIMP-3 and MMP mRNA
expression, chondrocytes were pretreated with curcumin for 30 min
followed by OSM treatment for 20 h, and total RNA was subjected to
Northern blot analysis. Curcumin, at a concentration of 15 µM,
completely (bovine chondrocytes) or partially (human chondrocytes)
inhibited MMP-1, -3, -13, and TIMP-3 induction by OSM without affecting
28S rRNA levels (Fig. 8). Thus, curcumin
abolished the enhancement of TIMP-3 and MMP expression by OSM in a
manner similar to that observed for the JAK3 inhibitor (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chondrocytes have an essential role in regulating the MMP-TIMP balance
and their primary function to maintain cartilage ECM integrity is
significantly altered in arthritis due to changed composition of growth
factors and cytokines (1, 42). The OSM-stimulated increase
of all of the major MMPs implicated in cartilage degradation (MMP-1,
-3, and -13) in both human and bovine chondrocytes suggests common
signaling mechanisms and supports a catabolic role for OSM (29, 30, 33, 34). OSM further enhances IL-1-induced MMP-13 in human
chondrosarcoma cells (43) and collagen degradation in
bovine nasal cartilage (29). Induction of MMPs in
chondrocytes differs from human synovial fibroblasts where MMP-1, but
not MMP-3, was induced by OSM (33), suggesting cell
type-specific regulation. MMP-1 and -3 induction by IL-1 and TNF-
was also enhanced by OSM in astrocytes (44). Intriguingly,
TIMP-3 induction by OSM in bovine but not in human chondrocytes may be
due to differences in the TIMP-3 promoter composition of two species
rather than lack of OSM receptor, as human chondrocytes do respond to
OSM by inducing MMPs. Induction of MMPs by OSM and noninduction of
TIMP-3 could tip the enzyme-inhibitor balance in favor of degradation.
In synovial fibroblasts, OSM inhibited the basal and IL-1-induced
TIMP-3 RNA (45). TIMP-3 was also not induced by OSM in
human astrocytes and dermal fibroblasts (44). OSM and
related cytokines, IL-6 and IL-11, do induce TIMP-1 in human
chondrocytes and in synovial/lung fibroblasts (31, 46, 47, 48). OSM, IL-6, and LIF expressed in cartilage and
rheumatoid synovium (27, 28) could contribute to the
pathogenesis of arthritis by altering TIMP-1, TIMP-3, and MMP gene
expression.
The IL-6-type cytokines, including OSM, are known to induce the JAK/STAT signaling cascade in other systems (35), but this has not been shown in chondrocytes. Our demonstration of OSM-triggered phosphorylation of JAK1, JAK2, JAK3, and STAT1 in primary human femoral head chondrocytes suggests that these cells could be a source of activated STAT1 and STAT3 found in the synovial fluid and fibroblasts of patients with RA (49, 50). The JAKs family consists of JAK1, JAK2, JAK3, and Tyk2 (51). In osteoblasts, OSM induced a rapid but transient tyrosine phosphorylation of JAK1, JAK2, and Tyk2 (52). Blockade of OSM-activated STAT1 tyrosine phosphorylation by JAK3, but not by the JAK2-specific inhibitor in our study, suggests that JAK3 (and possibly JAK1) and not JAK2 may be mediating STAT1 activation and OSM signal transduction in chondrocytes. This notion was further supported by the abolishment of downstream STAT1 activation, its DNA-binding activity, and TIMP-3 and MMP RNA induction by the JAK3 and not by the JAK2 inhibitor. These results suggest the pivotal role of the JAK/STAT pathway in OSM signaling leading to TIMP-3 and MMP expression. Activation of JAKs is followed by phosphorylation of the STAT family whose homo- or heterodimers in turn are translocated to the nucleus to activate their target genes (35, 36, 37) such as TIMPs and MMPs. Although this is the first report for induction of JAK/STAT pathway in chondrocytes, STAT1 DNA-binding activity induced by OSM was previously shown in synovial lining cells where OSM inhibits IL-1-induced TIMP-3 expression (45). Maximal activation of the transcriptional capacity of STAT1 also required serine phosphorylation (53, 54) by ERK2 (55). In human primary astrocytes, Raf/ERK and JAK/STAT pathways acted synergistically to achieve maximal induction of MMP-1 expression driven by OSM (44). This is consistent with the activation of all three subclasses of MAPKs in this study and previously shown inhibition of OSM-induced TIMP-3 and MMP-1, -3, and -13 in chondrocytes by the specific MAPK/ERK kinase (kinases upstream of ERKs) inhibitor PD98059 (Ref. 3 4 and our unpublished data). Together, these results strongly support the requirement of JAK/STAT, ERK, p38, and JNK pathways and possible interaction of these cascades in the OSM-enhanced TIMP-3 and MMP gene expression.
Curcumin (diferuloylmethane), a dietary pigment from Curcuma
longa, gives unique flavor and yellow color to curry and is a
pharmacologically safe compound. Its anticarcinogenic activities are
due to inhibition of tumor initiation and promotion by phorbol ester
(56, 57). Curcumin inhibits JNK, c-Jun, and NF-
B
activation by PMA or TNF- treatment (58, 59, 60, 61) and has
strong anti-inflammatory and antioxidant properties
(62). These therapeutic effects are believed to be due to
its abilities to block MAPK kinase kinase in the JNK signaling pathway
in Jurkat T cells, c-Jun expression in 3T3 cells, and IB kinase in
the NF-B activation pathway induced by diverse agents
(58, 59, 60, 61, 62). In agreement with these studies (58, 59), curcumin also effectively inhibits JNK activation in human
and bovine chondrocytes. Here, we show for the first time that curcumin
also interferes with the JAK/STAT signaling pathway specifically by
inhibition of OSM-induced STAT1 Tyr701
phosphorylation in chondrocytes, without blocking ERK, p38, JAK1, JAK2,
and JAK3 activation. The Raf/ERK pathway activation by IFN and OSM was
shown to be JAK1 dependent (63). Since curcumin did not
inhibit several of the OSM-induced activations, it displayed a
considerable degree of selectivity. STAT1 phosphorylation and
homodimerization leads to binding with its cognate DNA in the promoters
of the target genes (36, 51, 53). EMSA experiments showed
that indeed curcumin also prevented the binding of STAT1 to its target
DNA sequence. Curcumin mimicked the effects of the specific JAK3
inhibitor, which also blocked OSM-activated STAT1 phosphorylation and
its DNA-binding activity. In contrast, AG490, which does not block
STAT1 phosphorylation, failed to inhibit STAT1 DNA binding. Thus, our
study demonstrated that OSM-stimulated JAK3-mediated
Tyr701 phosphorylation of STAT1 plays a crucial
role in the transduction of this signal and curcumin has a hitherto
unknown and novel activity of specifically inhibiting STAT1
activation.
Binding of STAT1 homodimers to its cognate DNA element usually results in transcriptional up-regulation of the target genes (36, 51, 52, 53). Interestingly, by inhibiting upstream STAT1 phosphorylation and by preventing its ability to bind DNA, curcumin suppressed downstream TIMP-3, MMP-1, MMP-3, and MMP-13 gene up-regulation by OSM in chondrocytes. These results along with the analogous inhibition of TIMP-3 and MMPs by the JAK3 inhibitor further support curcumin as an effective inhibitor of the JAK/STAT pathway and implicate this pathway in the induction of these genes by OSM. The most likely target sequence of JAK/STAT signaling is the OSM-responsive element which encompasses STAT and AP-1 binding sites in the human MMP-1 and TIMP-1 and rat TIMP-1 gene promoters. For achieving maximal response by OSM, cooperation and cross-talk between STAT and AP-1 elements and the respective signaling cascades are needed via ERK2-MAPK-mediated STAT1 phosphorylation at Ser727 (44, 64). Consistent with the AP-1 involvement, OSM transiently increases early response genes such as c-fos which cooperates with the Ets and Sp1 factors to maximally induce transcription from the murine TIMP-1 promoter in hepatoma cells (65). Similar sequences may be present in the bovine and human MMP-3 and MMP-13 and possibly bovine TIMP-3 promoters which remain to be characterized. The human MMP-3 and MMP-13 promoters do contain AP-1 and Ets binding, PEA3 elements in close proximity (2), and PEA3 has sequence similarity with STAT binding sites (65). The human TIMP-3 promoter contains several Sp1 binding sites but no obvious STAT1 or STAT3 binding sites (45), which is consistent with lack of its induction by OSM in human chondrocytes. Abrogation of all of the OSM target genes, MMP-1, MMP-3, MMP-13, and TIMP-3 by the JAK3 inhibitor and curcumin suggest that common signaling or regulatory steps may be impaired, which seems to be STAT1 phosphorylation in the JAK/STAT and JNK activation in the MAPK pathway.
The ability of curcumin (and JAK3 inhibitor) to block OSM-induced
JAK/STAT and JNK pathways and MMPs may have potentially several
therapeutic applications. In arthritis, along with its known capacity
to inhibit AP-1 and NF-B transcription factors, curcumin may prevent
inflammation and protect cartilage from the OSM-, IL-1-, and TNF-
-stimulated degradation by MMPs. Furthermore, inhibition of fibroblast
growth factor 2-induced angiogenesis (a process where MMPs play an
important role) and MMP-9 by curcuminoids (66) can have
therapeutic benefits in RA and growth of tumors. Indeed, curcumin
inhibits cancer cell metastasis by inhibiting MMP-9 (67).
Curcumin can also be useful for blocking OSM-stimulated proinflammatory
responses in endothelial cells (68) and promotion of
vascular smooth muscle cell proliferation, the key events in
atherosclerosis.
In summary, we demonstrated that OSM activates JAK/STAT and MAPK (ERK, p38, and JNK) signaling pathways in primary chondrocytes, leading to induction of MMPs and TIMP-3 genes. Blocking STAT1 phosphorylation either by curcumin or by JAK3 inhibition results in abrogation of OSM-responsive MMP-1, MMP-3, MMP-13, and TIMP-3 genes. Therefore, JAK/STAT and MAPK pathways are essential in the transduction of OSM signal and regulation of MMP and TIMP-3 genes. The catabolic responses of OSM such as promotion of cartilage degradation in arthritis could possibly be blocked by the inhibitors of JAK/STAT and MAPK signaling cascades such as JAK3 inhibitor and curcumin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Muhammad Zafarullah, K-5255 Mailloux, Hôpital Notre-Dame du Centre Hospitalier de l’Université de Montréal, 1560 Sherbrooke est, Montreal, Quebec, Canada H2L 4M1.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; JAK, Janus kinase, JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase, MMP, matrix metalloproteinase; OSM, oncostatin M; TIMP-3, tissue inhibitor of metalloproteinase-3.
Received for publication July 17, 2000. Accepted for publication December 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 43:801.[Medline]
- and interferon 
-stimulated gene expression through STAT proteins. Science 269:1721.B is suppressed by curcumin (diferuloylmethane). J. Biol. Chem. 270:24995.B activation and proinflammatory gene expression by inhibiting inhibitory factor I-B kinase activity. J. Immunol. 163:3474. interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway. Mol. Cell. Biol. 17:3833.[Abstract]
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 S. Y. Eum, Y. W. Lee, B. Hennig, and M. Toborek Interplay between Epidermal Growth Factor Receptor and Janus Kinase 3 Regulates Polychlorinated Biphenyl-Induced Matrix Metalloproteinase-3 Expression and Transendothelial Migration of Tumor Cells Mol. Cancer Res., June 1, 2006; 4(6): 361 - 370. [Abstract] [Full Text] [PDF]
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 M. Cohen, S. Marchand-Adam, V. Lecon-Malas, J. Marchal-Somme, A. Boutten, G. Durand, B. Crestani, and M. Dehoux HGF synthesis in human lung fibroblasts is regulated by oncostatin M Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1097 - L1103. [Abstract] [Full Text] [PDF]
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L. C. Huber, O. Distler, I. Tarner, R. E. Gay, S. Gay, and T. Pap Synovial fibroblasts: key players in rheumatoid arthritis Rheumatology, June 1, 2006; 45(6): 669 - 675. [Abstract] [Full Text] [PDF]
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 R. Tabibiazar, R. A. Wagner, E. A. Ashley, J. Y. King, R. Ferrara, J. M. Spin, D. A. Sanan, B. Narasimhan, R. Tibshirani, P. S. Tsao, et al. Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease Physiol Genomics, July 14, 2005; 22(2): 213 - 226. [Abstract] [Full Text] [PDF]
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 K. S. Selander, L. Li, L. Watson, M. Merrell, H. Dahmen, P. C. Heinrich, G. Muller-Newen, and K. W. Harris Inhibition of gp130 Signaling in Breast Cancer Blocks Constitutive Activation of Stat3 and Inhibits in Vivo Malignancy Cancer Res., October 1, 2004; 64(19): 6924 - 6933. [Abstract] [Full Text] [PDF]
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 A. K. Behera, C. M. Thorpe, J. M. Kidder, W. Smith, E. Hildebrand, and L. T. Hu Borrelia burgdorferi-Induced Expression of Matrix Metalloproteinases from Human Chondrocytes Requires Mitogen-Activated Protein Kinase and Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathways Infect. Immun., May 1, 2004; 72(5): 2864 - 2871. [Abstract] [Full Text] [PDF]
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 J. Nightingale, S. Patel, N. Suzuki, R. Buxton, K.-i. Takagi, J. Suzuki, Y. Sumi, A. Imaizumi, R. M. Mason, and Z. Zhang Oncostatin M, a Cytokine Released by Activated Mononuclear Cells, Induces Epithelial Cell-Myofibroblast Transdifferentiation via Jak/Stat Pathway Activation J. Am. Soc. Nephrol., January 1, 2004; 15(1): 21 - 32. [Abstract] [Full Text] [PDF]
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H. Y. Kim, E. J. Park, E.-h. Joe, and I. Jou Curcumin Suppresses Janus Kinase-STAT Inflammatory Signaling through Activation of Src Homology 2 Domain-Containing Tyrosine Phosphatase 2 in Brain Microglia J. Immunol., December 1, 2003; 171(11): 6072 - 6079. [Abstract] [Full Text] [PDF]
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A. C. Bharti, N. Donato, and B. B. Aggarwal Curcumin (Diferuloylmethane) Inhibits Constitutive and IL-6-Inducible STAT3 Phosphorylation in Human Multiple Myeloma Cells J. Immunol., October 1, 2003; 171(7): 3863 - 3871. [Abstract] [Full Text] [PDF]
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G. S. Laszlo and N. M. Nathanson Src Family Kinase-independent Signal Transduction and Gene Induction by Leukemia Inhibitory Factor J. Biol. Chem., July 18, 2003; 278(30): 27750 - 27757. [Abstract] [Full Text] [PDF]
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A. D. Rowan, W. Hui, T. E. Cawston, and C. D. Richards Adenoviral Gene Transfer of Interleukin-1 in Combination with Oncostatin M Induces Significant Joint Damage in a Murine Model Am. J. Pathol., June 1, 2003; 162(6): 1975 - 1984. [Abstract] [Full Text] [PDF]
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 S. Sengul, C. Zwizinski, and V. Batuman Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells Am J Physiol Renal Physiol, June 1, 2003; 284(6): F1245 - F1254. [Abstract] [Full Text] [PDF]
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S. Dziennis and B. A. Habecker Cytokine Suppression of Dopamine-beta -hydroxylase by Extracellular Signal-regulated Kinase-dependent and -independent Pathways J. Biol. Chem., April 25, 2003; 278(18): 15897 - 15904. [Abstract] [Full Text] [PDF]
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J. K. S. Shum, J. A. Melendez, and J. J. Jeffrey Serotonin-induced MMP-13 Production Is Mediated via Phospholipase C, Protein Kinase C, and ERK1/2 in Rat Uterine Smooth Muscle Cells J. Biol. Chem., November 1, 2002; 277(45): 42830 - 42840. [Abstract] [Full Text] [PDF]
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 R. C. Humphreys, B. Bierie, L. Zhao, R. Raz, D. Levy, and L. Hennighausen Deletion of Stat3 Blocks Mammary Gland Involution and Extends Functional Competence of the Secretory Epithelium in the Absence of Lactogenic Stimuli Endocrinology, September 1, 2002; 143(9): 3641 - 3650. [Abstract] [Full Text] [PDF]
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C. Natarajan and J. J. Bright Curcumin Inhibits Experimental Allergic Encephalomyelitis by Blocking IL-12 Signaling Through Janus Kinase-STAT Pathway in T Lymphocytes J. Immunol., June 15, 2002; 168(12): 6506 - 6513. [Abstract] [Full Text] [PDF]
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