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Louis-Charles Simard Research Center, Centre Hospitalier de l’Université de Montréal (CHUM) Campus Notre-Dame and Department of Medicine, University of Montreal, 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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A family of tissue inhibitors of metalloproteinases (TIMPs), comprising TIMP-1, -2, -3, and -4, regulates the activities of MMPs by complexation and by maintaining an enzyme-inhibitor balance (5). TIMP-1 (6) and TIMP-2 (7) are the earlier members of the gene family, while TIMP-3 (8) and TIMP-4 (9) are the most recent. MMPs and TIMPs play vital roles during physiologic ECM turnover in animal development and aging. Pathologic turnover of ECM is believed to be a consequence of TIMPs-MMPs imbalance, as shown for the OA cartilage (10). Such imbalance is also implicated in retinal degeneration, periodontal diseases, atherosclerotic plaque rupture, and metastatic invasion of cancer cells. TIMPs have both growth-promoting and antimetastatic activities (11, 12).
TIMP-3, a 21-kDa ECM-associated protein from transformed chicken fibroblasts, is distinct yet related to other TIMPs (13, 14). It stimulates the proliferation of nontransformed cells (15). Human TIMP-3 cDNA was isolated from placenta, and its expression was shown in different tissues (8), such as fetal kidney (16) and breast tumors (17). Murine TIMP-3 is expressed in fibroblasts and animal tissues, and the protein is found in ECM only (18). TIMP-3 is up-regulated by mitogens at the G1 phase of cell cycle (19). Gene mutations of TIMP-3, a component of Bruch’s membrane of the eye (20), could interfere with inhibition of MMPs, possibly causing a retinal degenerative disease (21). TIMP-3 mutations can influence its ability to inhibit angiogenesis of the eye (22). By its presence in ECM, TIMP-3 blocks tumor growth (23), possibly by inhibiting angiogenesis (24). The extent of TIMP-3 inhibition of MMPs is similar to that of TIMP-1 (25). However, TIMP-3 and TIMP-2 are better inhibitors of MT1-MMP compared with TIMP-1 (26). Recent studies have related TIMP-3 expression with chondrocyte differentiation during mouse development (27, 28). We have observed that TIMP-3 and TIMP-1 RNA expression is increased by unknown signals in human OA synovial membranes (Su et al., unpublished observations), and the two genes are inducible in human and bovine chondrocytes by an inducer of matrix synthesis, TGF-ß (29, 30). Due to their potential for inhibiting cartilage resorption, studying the regulatory mechanisms of TIMPs by pathophysiologically relevant stimuli in cartilage could be of therapeutic value in arthritis.
Oncostatin M (OSM), a 28-kDa glycoprotein produced by activated
T-lymphocytes and monocytes, inhibits the growth of human melanoma
cells (31) and is a mitogen for normal fibroblasts and smooth muscle
cells. OSM belongs to the IL-6 family of cytokines, including leukemia
inhibitory factor (LIF), that share structural features, binding to the
receptor gp 130 and linkage of their genes (OSM and LIF) on the human
chromosome 22 (32). OSM was undetectable in the synovial fluid of OA
but was present in the RA patients, suggesting its role in joint
inflammation (33). OSM mRNA and protein were elevated in the human RA
vs OA synovial cells (34). Previous studies have shown that OSM and
other IL-6-type cytokines induced TIMP-1 but not MMP gene expression in
human lung and synovial fibroblasts and could influence matrix
degradation during chronic inflammation (35). A proinflammatory role
for OSM was suggested since it stimulates degradation of porcine
cartilage and, like IL-1 and TNF-
, inhibits proteoglycan synthesis
(36). IL-1 and OSM, in combination, promote bovine nasal cartilage
proteoglycan and collagen degradation (37). In contrast, OSM was
regarded as a cartilage protective cytokine due to its induction of
TIMP-1 in human chondrocytes (38). A recent study showed that, in
rheumatoid fibroblasts, OSM increased TIMP-1 and inhibited
IL-1ß-induced TIMP-3 RNA expression (39). Cartilage is a major target
tissue for pro- and antiinflammatory cytokines in arthritis, which
could influence the outcome of the disease by regulating a plethora of
genes. None of the previous studies have addressed regulation of the
recently described TIMP-3 gene by OSM in chondrocytes, which is a
unique cell type whose primary function is to maintain cartilage ECM
(40). Due to the contradictory role of OSM in joints, this issue is of
paramount biologic and pathophysiologic significance. The aims of the
present study were to assess the role of OSM in cartilage by
investigating its impact on TIMP-3 gene expression and the possible
mechanisms of this response in articular chondrocytes. Here, we
demonstrate for the first time, TIMP-3 up-regulation by OSM and
inhibition by dexamethasone in primary bovine chondrocyte model of
cartilage research and show dependence of the response on new protein
and RNA synthesis. We also demonstrate suppression of TIMP-3 RNA
expression by genistein, herbimycin A, and PD98059, suggesting the
involvement of tyrosine kinase and MAPK pathways in OSM signaling.
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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, antibiotic-antimycotic agents, trypan blue, and agarose were from Life Technologies (Burlington, Ontario, Canada). Plastiware was from Nalge Nunc (Naperville, IL). Human recombinant oncostatin M and human platelet TGF-ß, which were reconstituted as recommended, were from R&D Systems (Minneapolis, MN). Pronase, clostridial collagenase type II, actinomycin-D, and dexamethasone were from Sigma (St. Louis, MO). Cycloheximide was acquired from U.S. Biochemical (Cleveland, OH). Protein kinase inhibitors, genistein, herbimycin A, and PD098059 were from Calbiochem (San Diego, CA). Zeta-probe nylon membrane was from Bio-Rad (Mississauga, Ontario, Canada). RNA probe labeling kits were from Promega (Madison, WI). [32P]CTP was from Dupont-NEN (Boston, MA). Restriction endonucleases were from Pharmacia Biotech (Baie d’urfé, Quebec, Canada) and Boehringer Mannheim (Laval, Quebec, Canada). Other common reagents were from Fisher Scientific (Montreal, Quebec, Canada).
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. The cartilage-containing bones were dipped briefly in 1% proviodine (Rougier, Chambly, Quebec, Canada) for sterilization and washed extensively with 0.9% NaCl. The slices of cartilage were dissected out, kept for 1 to 2 h at 4°C in 5x antibiotic-antimycotic solution, and washed five times with large volumes of PBS containing 5x penicillin-streptomycin and 1x fungizone (Life Technologies). Chondrocytes were released from bovine cartilage by digestion with pronase (1 mg/ml) for 90 min and collagenase (Sigma type II) for 12 h in DMEM at 37°C. Viability by trypan blue exclusion test was about 80%. The cells were pelleted and washed three 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). Before different treatments, cells were kept in serum-free DMEM for 24 h. The potential inhibitors were added 30 min before the OSM.
RNA extraction and Northern hybridization analysis
Total RNA from primary cultures of chondrocytes was extracted by
the acid-guanidinium procedure (41), and 5-µg aliquots were analyzed
by fractionation in 1.2% formaldehyde-agarose gels, transferred to
Zeta-probe membranes, and hybridized as previously described (42). The
quality and quantity of applied RNA were verified visually by ethidium
bromide staining and photography of 28S and 18S ribosomal RNA bands.
The RNA was electroblotted onto Zeta-Probe nylone membrane (Bio-Rad)
using a Bio-Rad Transblot in the presence of 1x TAE (Tris acetate
EDTA) buffer at a current of 500 mAmp for 12 h. Complete transfer
was ascertained by ethidium bromide staining of the gel where no
ribosomal RNA bands were visible. Northern blot analysis of RNA was
performed with a bovine TIMP-3 cDNA probe (29). This probe was a
2.0-kbp EcoRI-EcoRI cDNA fragment cloned in the
plasmid pGEM4-Z (Promega), which was linearized with NarI
and RNA probe synthesized with T7 polymerase according to the protocols
of Promega. The human 28S ribosomal RNA probe (from American Type
Culture Collection (ATCC), Manassas, VA) was a 1.5-kb cDNA fragment
cloned in pBluescript SK-. This vector was digested with
XbaI, and a probe was synthesized using T7 polymerase. For
Figure 1
, membranes were also hybridized
sequentially with the human collagenase-1 (clone pSP64) and stromelysin
(from ATCC and Dr. Richard Breathnach, Nantes, France respectively) RNA
probes, which cross-hybridize with the corresponding bovine
transcripts. All probes were routinely labeled to high-specific
activity (1 x 108 cpm/µg) with
[-32P]CTP (3000 Ci/mmol). The prehybridization,
hybridization, and posthybridization wash conditions were described
earlier (42). The final stringent wash was in 0.1% SET.
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For the analysis of the TIMP-3 protein, chondrocytes treated with or without OSM were washed twice with PBS, lysed in 100 µl of the lysis buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, and 1% Triton X-100), and sonicated for 15 s; and the protein was quantified by the Bio-Rad protein assay system. Aliquots of 40-µg protein were adjusted to 2% SDS and 10% mercaptoethanol and boiled for 5 min. After centrifugation and addition of bromophenol blue, the samples were electrophoresed on a 12% mini SDS-PAGE gel along with the size markers and transferred to nitrocellulose membranes by electroblotting in a buffer consisting of 12.5 mM Tris (pH 8.0) and 100 mM glycine, at 250 mA using the Bio-Rad system. The blots were washed for 5 min in TBS (20 mM Tris-HCl (pH 7.6) and 135 mM NaCl) and blocked with 3% nonfat Carnation milk in TBS and 0.05% Tween 20 for 1 to 3 h at room temperature. Following a wash with TBS for 10 min, the blots were incubated overnight with gentle agitation at 4°C with 5 µg/ml of the human TIMP-3 mAb (20) (Ab-1, clone 136-13H4 from Calbiochem) in TBS and 0.5% milk. The membranes were washed 3 times with TBS for 5 min each and incubated with TBS and 0.5% milk for 10 min, followed by incubation with anti-mouse secondary IgG-POD Fab fragment (peroxidase) (Boehringer Mannheim) for 1 h and 3 washes for 5 min each. The protein bands were revealed with Boehringer Mannheim chemiluminescence detection system and exposure to x-ray film for 3 min.
Protease substrate gel electrophoresis
Cells were exposed to OSM for 48 h, and 30 µl of the
medium was either used as such or treated with 1 mM
4-aminophenylmercuric acetate (APMA), mixed with 4x sample buffer
(0.25 M Tris-HCl (pH 6.8), 10% SDS, 4% sucrose, and 0.1% bromophenol
blue), and applied to 10% SDS-PAGE containing 1 mg/ml gelatin or
-casein (Sigma). Following electrophoresis under nonreducing
conditions (except m.w. standards), the gels were washed twice, 30 min
each in 2.5% Triton X-100 (V/V) and incubated overnight in 50 mM
Tris-HCl (pH 8.0) containing 5 mM CaCl2 and 1 µM
ZnCl2 and 0.02% NaN3 at 37°C. Following
staining with 0.1% Coomassie blue in water:methanol:acetic acid
(5:4:1) for 10 min and destaining, gelatinolytic or caseinolytic
activities were detected by the zones of lysis in the respective
molecular mass region and photographed.
All the experiments reported in this paper were performed at least twice with different batches of cells, and the results were highly reproducible.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether OSM was able to regulate TIMP-3 gene in
mammalian chondrocytes, primary bovine cells in two separate
experiments were kept in serum-deprived medium for 24 h and then
exposed for an other 24 h in the same medium to different
concentrations (0.1–50 ng/ml) of recombinant human OSM. Northern
hybridization analysis of RNA with the bovine TIMP-3 and 28S rRNA
probes, respectively, revealed a potent dose-dependent induction of the
three TIMP-3 transcripts relative to the mostly even 28S RNA levels
(Fig. 1
). The maximal induction was with the doses between 10 to 50
ng/ml. Sequential hybridization with two metalloproteinase probes
revealed that stromelysin-1 and collagenase-1 RNAs were also moderately
inducible with OSM (6 days film exposure compared with 18 h for
TIMP-3).
To examine whether the RNA induction was followed by the respective
protein synthesis, total cellular extracts or the media from the
untreated and OSM-treated cells were analyzed for TIMP-3 protein
and MMP activities, respectively. Western immunoblot analysis with a
mouse anti-human TIMP-3 mAb (20) demonstrated a dose-dependent
TIMP-3 induction as revealed by a 24-kDa band equivalent to
nonglycosylated bovine TIMP-3 (Fig.
2A). Zymographic analysis
showed a similar pattern of caseinolytic and gelatinolytic activities
induction corresponding to stromelysin (Fig. 2B),
collagenases, and gelatinases (Fig. 2C). Therefore, TIMP-3
and MMP genes are clearly responsive to OSM, and their RNAs and
proteins are inducible by this cytokine in articular chondrocytes.
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TGF-ß is an inducer of cartilage ECM, TIMP-1 (43), and TIMP-3
(29), as well as an inhibitor of MMPs (44). To examine the effect of
the two factors in combination, chondrocytes were subjected to
individual or combined treatments. In two separate series of cells,
TGF-ß was a more potent inducer of TIMP-3 message and protein (latter
not shown) compared with OSM, and the combined treatments of TGF-ß
and OSM did not further increase the levels of induction relative to
mostly constant levels of 28S rRNA. Thus, there are no synergistic or
additive effects of the two factors on TIMP-3 gene expression (Fig. 3).
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To investigate the mechanism of TIMP-3 RNA enhancement by OSM, the
time-course of induction was determined by maintaining chondrocytes in
serum-free medium or by exposing them to OSM (10 ng/ml) for different
time periods. Northern hybridization analysis revealed that the TIMP-3
message induction occurred rapidly within 3 h, peaked at 14
h, remained elevated for 24 h, and declined drastically by 36
h (Fig. 4). The 28S rRNA levels remained
relatively consistent. Thus, TIMP-3 gene is regulated by OSM in a
time-dependent fashion.
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To gain insight into the mechanism of TIMP-3 mRNA up-regulation by
OSM, the effect of protein synthesis inhibitor cycloheximide (CHX) and
that of transcriptional inhibitor actinomycin-D (Act-D) on induction
was studied by individual and combined treatments. OSM induced TIMP-3
mRNA while CHX (10 µg/ml) and Act-D (1 µg/ml), added 30 min before
OSM, strongly inhibited this induction. The levels of 28S rRNA were
invariable and demonstrated even application of RNA (Fig. 5). Thus, OSM enhancement of TIMP-3
message is dependent on new protein synthesis and transcription.
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To investigate whether TIMP-3 augmentation was due to increased
RNA stability by OSM, TIMP-3 RNA was first induced in chondrocytes by a
24-h treatment with this cytokine, and medium was replaced with Act-D
only or Act-D and OSM. Treatments were stopped at different time
points, and TIMP-3 RNA was analyzed to follow the RNA decay time
course. As shown in Figure 6, stability
of the TIMP-3 RNA was unaffected by OSM. The constant levels of 28S
rRNA were not affected by the treatments and also demonstrated equal
application. Therefore OSM-stimulated enhancement of TIMP-3 message is
not due to its increased stability.
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We have demonstrated that the synthetic glucocorticoid,
dexamethasone (Dex) inhibits the basal and TGF-ß-induced TIMP-3 RNA
expression (29). To examine the effect of Dex on the OSM stimulation of
TIMP-3 mRNA expression, chondrocytes were exposed in FCS-free medium to
the ethanol vehicle (for Dex), OSM and Dex individually, or pretreated
with Dex (1 µg/ml) for 30 min followed by OSM treatement for 24
h. Dex further reduced the basal TIMP-3 RNA levels and strongly
inhibited its induction by OSM without affecting the 28S rRNA levels
(Fig. 7). Thus Dex potently
down-regulates OSM-induced TIMP-3 gene expression.
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To investigate the mechanisms of OSM signaling leading to TIMP-3
RNA increase, chondrocytes were treated with vehicles (Control), OSM,
genistein (50 and 100 µM), or herbimycin A (5 and 15 µM) (tyrosine
kinase inhibitors), either individually or in combination (pretreatment
with the inhibitors for 30 min) for 24 h. Both inhibitors
dose-dependently suppressed the OSM induction of TIMP-3 gene expression
while the 28S rRNA levels were unaffected (Fig. 8). A recently developed, very specific
inhibitor of MAPK kinase, PD98059 (45), at a 30-µM dose, partially
inhibited the OSM action (not shown), while the doses of 60 and 100
µM strongly diminished this induction to basal levels without
influencing the constant levels of 28S RNA (Fig. 9). Two separate batches of cells for
each of the above experiments gave similar results.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TIMP-3 induction in bovine chondrocytes differs from a study in human rheumatoid cells where IL-1ß-induced TIMP-3 RNA was inhibited by OSM, suggesting the latter’s antiinflammatory role (39). This may be due to cell-type-specific differential regulation. OSM and TIMP-3 may have different roles in cartilage and synovium. In cartilage, TIMP-3 RNA and protein increase by OSM may have a protective role, while, in synovial linings, TIMP-3 increase (Su et al., unpublished results) may be related to inflammation and cell proliferation, since it is a cell-cycle-associated gene (19). OSM and related cytokines IL-6 and IL-11 also induce TIMP-1 in human chondrocytes and in lung/synovial fibroblasts (35, 38, 46, 47). However our results suggest that, besides TIMP-1 and TIMP-3, collagenase-1 and stromelysin-1 are also coordinately regulated by OSM. Collagenase-1, but not stromelysin-1, was recently shown to be induced by OSM in human synovial fibroblasts (48). OSM, IL-6 (49), and LIF (50) are expressed in cartilage and synovium and, through autocrine mechanisms, could influence joint metabolism by regulating TIMP-3, TIMP-1, and MMPs.
The time course suggests that OSM rapidly induces TIMP-3 gene in chondrocytes. Decline in the response by 36 h may be due to degradation of the exogenous OSM. Therefore, normal chondrocytes have OSM-signaling components that lead to the TIMP-3 response. Deficiency of this response in the RA and OA chondrocytes, which are hyporesponsive to TGF-ß and insulin-like growth factor (IGF)-I (51), is not known. The proposed cartilage protection by OSM may be due to its induction of TIMP-3 and TIMP-1, which could block the degradative MMPs. However, increased OSM and TIMPs in arthritic tissues may not protect individuals from arthritic damage, and their enhancement may indicate attempted repair. Simultaneous induction of MMPs by OSM questions its protective function (36) and suggests a role in cartilage remodeling.
Although OSM and TGF-ß induced TIMP-3 RNA expression individually, there was no synergy or additive effect between them. TGF-ß was a more potent inducer of TIMP-3 protein than OSM, suggesting different mechanisms of induction; indeed, both factors suppressed mouse B cell hybridoma proliferation through distinct mechanisms (52). Interestingly, both OSM and TGF-ß also induce TIMP-1 in human chondrocytes (30, 38).
TIMP-3 inhibition by CHX and Act-D suggests the need for transcription and synthesis of intermediate products for induction by OSM. Interestingly, TGF-ß augmentation of TIMP-3 RNA can also be blocked with these reagents, suggesting partial similarities in the induction mechanisms. OSM induces TIMP-3 RNA expression at the level of transcription, a view supported by the lack of increased TIMP-3 RNA stability by this agent. The putative intermediary gene products inhibited by CHX and Act-D may be the tyrosine kinases (Janus kinases, JAKs), STATs, and the immediate-early response genes. These conclusions were supported by Dex suppression, which prevents the binding of c-jun to its target sequences in the promoter by DNA-free interactions with the glucocorticoid receptor (53). Dex may have blocked STATs in a similar fashion. Dex also suppresses the basal and TGF-ß induction of TIMP-3 RNA in bovine and human chondrocytes (29). Thus, glucocorticoids can block the action of a variety of pro- and antiinflammatory cytokines. The phospholipase gene induction by OSM in human hepatoma cells was also inhibited by Dex (54). However, in Kaposi’s sarcoma cells, Dex stimulates gp130-mediated growth by increasing the accumulation of tyrosine phosphorylated STAT3 (55). Thus, Dex may act differentially on OSM action in different cell types. If OSM contributes to cartilage resorption (36, 37), glucocorticoids may suppress its actions in chondrocytes.
The suppression of OSM-stimulated TIMP-3 gene expression by genistein and herbimycin A suggests the implication of tyrosine kinases. Genistein also inhibits the induction of low density lipoprotein and Egr-1 transcription factor by OSM (56). One possible site of genistein action is OSM activation of tyrosine kinases, Janus kinases (Tyk-1, Jak-1, Jak-2) (57) that phosphorylate STAT-1 and STAT-3 and whose homo- or heterodimers (58) in turn are translocated to the nucleus to activate the target genes, such as TIMP-3 and TIMP-1. The JAKs/STATs are involved in the IFN, IL-6, OSM, and LIF signaling (59). In other cell types, there are certain steps in OSM signaling that involve tyrosine phosphorylation. Since MAPK (possibly ERK-2) further activates STATs, the tyrosine phosphorylation steps in the MAPK pathway may also have been inhibited by herbimycin A and genistein. Another interaction between the two pathways may be activation of Raf by JAKs (60). Indeed, genistein inhibits tyrosine phosphorylation and MAPK activation in Kaposi’s sarcoma cells (61).
Inhibition of TIMP-3 RNA expression by the MAPK kinase-specific
inhibitor strongly supports the involvement of the MAPK pathway in OSM
signaling. Blocking of any upstream step during MAPK activation of AP-1
and OSM activation of STATs may inhibit TIMP-3. The involvement of AP-1
as targets of OSM signaling is supported by the down-regulation of
TIMP-3 message induction by curcumin, a specific AP-1 inhibitor (our
unpublished results). Recent studies suggest that phosphorylation of a
serine residue in Stat-1 by MAPKs is needed for IFN-induction of the
target genes. OSM-responsive elements are found in the rat TIMP-1
promoter (62), and there is cross-talk between the MAPK and JAK-STAT
pathways (59, 63).
In summary, we have demonstrated coordinate OSM up-regulation of TIMP-3, MMP-1, and MMP-3 RNA and protein, lack of synergy between TGF-ß and OSM, de novo transcription and protein synthesis dependence of TIMP-3 gene induction, inhibition by Dex, and the involvement of tyrosine and MAP kinase pathways. Thus, in normal mammalian chondrocytes, OSM may contribute to cartilage ECM remodeling. Due to MMP inhibition and potent induction of TIMPs, TGF-ß may be superior to OSM for cartilage protection.
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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, CHUM Campus Notre-Dame, 1560 Sherbrooke est, Montreal, Québec, Canada H2L 4 M1. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; Act-D, actinomycin-D; AP-1, activator protein-1; CHX, cycloheximide; Dex, dexamethasone; LIF, leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; OSM, oncostatin M; OA, osteoarthritis; TIMP, tissue inhibitor of metalloproteinase; APMA, 4-aminophenylmercuric acetate; ECM, extracellular matrix.
Received for publication December 22, 1997. Accepted for publication June 25, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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