120

The Regulation of MMP

S

and TIMP

S

in Cartilage Turnover

TIM CAWSTON,

a

CARON BILLINGTON, CATHERINE CLEAVER, SARAH ELLIOTT, WANG HUI, PAUL KOSHY, BILL SHINGLETON, AND ANDREW ROWAN

Department of Rheumatology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, England

ABSTRACT: The treatment of cartilage with mediators initiates the break-down of proteoglycan followed by collagen. This is accompanied by the modu-lation of different proteinases and inhibitors that include members of theMMP family and TIMPs. We have evidence that a chondrocyte membrane-associated metalloproteinase cleaves aggrecan. This activity is rapidly inducedafter stimulation with IL-1 and OSM and is not inhibited by TIMPs-l and -2but is inhibited by synthetic MMP inhibitors. This same combination of cytok-ines also upregulates the collagenases with the subsequent release of collagenfragments, and there is a close correlation between the amount of collagen re-leased and collagenase activity produced. Collagen release can be preventedafter treatment with specific inhibitors of MAP kinases, inhibitors of MMPtranscription, synthetic metalloproteinase inhibitors, TIMPs and treatment ofcartilage with agents that upregulate TIMPs. The results from bovine cartilageculture models show that collagen release occurs when TIMP levels are low,collagenases are upregulated and then subsequently activated.

The matrix metalloproteinases (MMPs) are a unique family of enzymes that in con-cert can degrade all the components of the extracellular matrix.

1

These potent en-zymes are made in a proenzyme form, and activation occurs after leaving the cell.Extracellular activity is also controlled by specific inhibitors called tissue inhibitorof metalloproteinases (TIMPs),

2

which bind to the active forms of the enzyme, form-ing 1:1 complexes, and block activity. The inflammatory cytokine interleukin-1(IL-1) can induce resorption of cartilage

in vitro

3

and

in vivo

when injected into rab-bit joints

4

; it is also found in joint fluids from patients with rheumatoid arthritis.

5

IL-1 is known to stimulate the production of the MMPs, collagenase-1, andstromelysin-1 from human synovial cells and chondrocytes.

6

Raised levels of theseenzymes have been localized at both the mRNA and protein level within diseasedcartilage from different rheumatic diseases.

7

Consequently, the MMPs are a validtherapeutic target for inhibition in the rheumatic diseases.

8

A number of studies have shown that IL-1-stimulated cartilage model systemscan be used to test the effectiveness of proteinase inhibitors. The release of gly-cosaminoglycan (GAG) fragments can be prevented by the addition of low molecu-lar weight synthetic inhibitors,

9

but not by the addition of TIMPs.

10

However, while

a

Address for telecommunication: Phone, 0191 222 5363; fax, 0191 222 5455 e-mail,T.E.Cawston@ncl.ac.uk

121CAWSTON

et al.

: MMP

S

& TIMP

S

IN CARTILAGE TURNOVER

the release of GAG fragments from cartilage appears to be rapid in response toIL-1,

4,11

it is also quickly resynthesized by chondrocytes.

12

In contrast, the releaseof collagen from cartilage is much slower, is less reproducible, and appears to be ir-reversible as resynthesis is difficult to achieve.

13

In this study we have examined the release of both collagen and proteoglycanfragments from resorbing cartilage in response to a combination of IL-1 and oncos-tatin M (OSM) and we have studied different agents that block this release by thedownregulation of MMPs.

MATERIALS AND METHODS

Chemicals were obtained from the following suppliers: IL-1

α

was a generous giftfrom GlaxoWellcome, Stevenage, UK. OSM and interleukin-4 (IL-4) was obtainedfrom R&D Systems, Abingdon, UK. All other chemicals/biochemicals were analyt-ical grade reagents obtained from Merck Ltd, Poole, UK or have been previously de-scribed.

10

Cartilage Degradation Assay

Control culture medium was Dulbecco’s modification of Eagle’s medium (DMEM)containing 25 mM HEPES supplemented with sodium bicarbonate (0.5 g/l),glutamine (2mM), streptomycin (100

µ

g/ml), penicillin (100 U/ml), and amphotericin(2.5

µ

g/ml). Cartilage slices were dissected from bovine nasal septum cartilage anddiscs (2

×

2 mm) cut and washed twice in HBSS. Three discs were incubated at 37°Cin control medium (600

µ

l) in a 24-well plate for 24 hours for stabilization. Controlmedium (600

µ

l) with or without cytokines was added and the plate incubated at 37°Cfor 7 days. The supernates were harvested and replaced with fresh medium containingidentical test reagents to day 0. The experiment was continued for a further 7 days andday-7 and -14 supernates were stored at

−

20°C until assay. For experiments investi-gating the prevention of collagen degradation then IL-4 and transforming growth fac-tor-

β

(TGF-

β

) were added to the culture medium at day 0 and day 7 at theconcentrations shown. For human cartilage assays articular cartilage was dissectedfrom joint replacement samples, and three pieces of cartilage were incubated andtreated as described for bovine cartilage. In order to determine the total GAG andOHPro content of the cartilage fragments, the remaining cartilage was digested withpapain (2.5

µ

g/ml) in 0.1 M phosphate buffer, pH 6.5, containing 5 mM EDTA and5 mM cysteine hydrochloride, incubating at 65°C until digestion was complete(16 hours).

Proteoglycan Degradation

Media samples and papain digests were assayed for sulfated GAG (as a measureof proteoglycan release) using a modification of the 1,9-dimethylmethylene blue dyebinding assay.

14

Sample or standard (40

µ

l) was mixed with dye reagent (250

µ

l),prepared as described in the well of a microtiter plate, and the absorbance at 525 nmdetermined immediately. Chondroitin sulfate from shark fin (5–40

µ

g/ml) was used

122 ANNALS NEW YORK ACADEMY OF SCIENCES

as a standard. The complex formed with 1,9-dimethylmethylene blue decreases ab-sorbance at 525 nm.

Collagen Degradation

Hydroxyproline release was assayed (as a measure of collagen degradation) usinga microtiter plate modification of the method of Bergman and Loxley.

15

ChloramineT (7% [w/v]) was diluted 1:4 in acetate-citrate buffer (57 g sodium acetate, 37.5 gtri-sodium citrate, 5.5 g citrate acid, 385 ml propan-2-ol /liter water).

P

-dimethy-laminobenzaldehyde (DAB; 20 g in 30 ml 60% perchloric acid) was diluted 1:3 inpropan-2-ol. Specimens were hydrolyzed in 6 M HCl for 20 hours at 105°C and theacid removed on a centrifugal evaporator. The residue was dissolved in water and40

µ

l sample or standard (hydroxyproline; 5–30

µ

g/ml) added to microtiter plate to-gether with chloramine T reagent (25

µ

l) and then DAB reagent (150

µ

l) after 4 min.The plate was covered and heated to 60°C for 35 min and the absorbance at 560 nmwas determined.

Immortalized Chondrocytes

T/C28a4 chondrocytes

16

were grown to 70% confluence in DMEM +10% fetalcalf serum (FCS). Medium was replaced with DMEM supplemented with 1% acid-treated FCS (ATFCS) and incubated for 24 hours. Medium was replaced withDMEM

+

ATFCS containing the following agents: IL-1 (1 ng/ml), OSM (10 ng/ml),retinoic acid (10

−

6

M), IL-4 (10 ng/ml), or TGF-

β

(2 ng/ml). Cells were incubatedwith the cytokines, alone or in combination, for 24 hours. Medium was harvestedand stored at

−

20°C until assayed.

Enzyme and Inhibitor Assays

For experiments using human cells, TIMP-1 and collagenase (MMP-1) were as-sayed by specific ELISAs.

17

For experiments using bovine cartilage, collagenase ac-tivity was determined by the diffuse fibril assay

10

using

3

H-acetylated collagen.Inhibitory activity was assayed by the addition of samples to a known amount of ac-tive collagenase in the diffuse fibril assay. Aggrecanase activity was assayed as de-scribed.

18

Preparation of Aggrecanase

Bovine nasal chondrocytes were prepared and harvested at passage 2 after stim-ulation with IL-1 and OSM and a membrane fraction prepared.

18

The plasma mem-brane fraction and a sample of the cytosolic fraction was incubated with purifiedbovine aggrecan overnight at 37°C.

18

SDS final sample buffer was added and incu-bated at 100°C for 3 minutes and samples were separated by SDS-PAGE. After West-ern blotting onto nitrocellulose the degraded aggrecan fragments were detectedusing the antibody T767 as described.

18

123CAWSTON

et al.

: MMP

S

& TIMP

S

IN CARTILAGE TURNOVER

RESULTS

Aggrecan Degradation in Human Cartilage

Human cartilage was treated with IL-1 (5 ng/ml), OSM (50 ng/ml), and a mixtureof IL-1

+

OSM (5 ng/ml and 50 ng/ml) in qaudruplicate wells. Medium was removedon day 7 and the amount of GAG measured. The % release of total GAG is shownafter treatment with media alone, IL-1, OSM, and IL-1+OSM. A marked increase inproteoglycan release is seen in response to IL-1 and OSM (F

IG

. 1).

Chondrocyte Membrane Aggrecanase

When chondrocyte membranes were prepared from cells treated for 24 hours withIL-1

+

OSM, a large amount of associated aggrecanase activity was detected on themembranes (F

IG

. 2). Little aggrecanase activity could be demonstrated in the cyto-solic fraction.

Collagen Release and the Stimulation of MMP-1 in Human Articular Cartilage

Assay of human articular cartilage culture medium (day 7, 14, and 21) for hy-droxyproline showed a similar increase in the release of collagen into the medium inresponse to IL-1

+

OSM with a cumulative total of 16.03

±

7.39% of the total col-lagen (data not shown). Assay of the medium for MMP-1 and TIMP-1 demonstratesthat the cumulative levels of MMP-1 released into the medium are increased by IL-1 alone but a large synergistic increase in the amount of MMP-1 protein was seen in

FIGURE 1. The effect of IL-1 in combination with OSM on the release of GAG frag-ments from human articular cartilage. Human articular cartilage was cultured in quadrupli-cate in 600 µL of medium alone, medium + 5 ng/ml IL-1, medium + 50 ng/ml OSM andmedium + 5 ng/ml IL-1 + 50 ng/ml OSM. Medium was removed on day 7 and replaced,removed at day 14 and replaced, and then harvested at day 21. The remaining cartilage wasdigested with papain and the levels of GAG released on day 7 were measured and expressedas a % of the total. Results are expressed as mean ± SD. *** = p < 0.001.

124 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. Detection of aggrecanase on bovine chondrocyte membranes after stimu-lation with IL-1 +OSM. Confluent bovine chondrocytes cultured with IL-1 +OSM for 24hours were harvested and lysed. Cell nuclei were removed by centrifugation and the super-natant spun at 33,000g for 1.5 hr. Fractions of the supernatant and the resuspended mem-brane pellet were assayed for aggrecanase activity. High levels of activity were associatedwith the membrane pellet.

FIGURE 3. The cumulative levels of MMP-1 and TIMP-1 released into the mediumfrom human articular cartilage treated with IL-1 +OSM. Human articular cartilage was cul-tured in quadruplicate in 600 µL of medium alone, medium + 5 ng/ml IL-1, medium + 50ng/ml OSM, and medium + 5 ng/ml IL-1 + 50 ng/ml OSM. Medium was removed on day7 and replaced, removed at day 14 and replaced, and then harvested at day 21. The levelsof MMP-1 and TIMP-1 were measured by ELISA at each time point and the total amountof each measured. IL-1 alone stimulated the release of MMP-1, and a large, synergistic in-crease in MMP-1 levels was seen in combination with OSM. Although OSM alone in-creased TIMP-1 levels, this effect was reversed when OSM was combined with IL-1. Theresults are expressed as a cumulative total mean ± SD released by day 21. ** = p < 0.01;* = p < 0.05.

125CAWSTON

et al.

: MMP

S

& TIMP

S

IN CARTILAGE TURNOVER

the wells treated with IL-1

+

OSM (F

IG

. 3). OSM alone increased TIMP-1 produc-tion, but TIMP-1 levels were markedly reduced when IL-1

+

OSM was present(F

IG

. 4). IL-1

+

OSM markedly increased aggrecanase activity on chondrocyte mem-branes and proteoglycan release from cartilage. Collagen release was also increasedwith raised levels of MMP-1 and lowered levels of TIMP-1 in the medium at day 14.

Control of TIMP-1 Production in Chondrocytes by Growth Factors and Retinoic Acid

T/C28a4 chondrocytes

16

were treated with a variety of growth factors and the lev-els of TIMP-1 measured by ELISA

17

after 24 hours. IL-1 (1 ng/ml) had no effect onTIMP-1 levels, whilst OSM (10 ng/ml), TGF-

β

(2 ng/ml), retinoic acid (10

–6

M) andIL-4 (10 ng/ml) stimulated the production of TIMP-1. Some increase in levels wereseen when these agents were combined. It was decided to test the effectiveness of

FIGURE 4. Upregulation of TIMP-1 levels by growth factors and retinoic acid. T/C28a4 chondrocytes were treated with medium alone, medium + IL-1 (1 ng/ml), OSM(10 ng/ml), retinoic acid (10–6M), IL-4 (10 ng/ml), TGFβ (2ng/ml), IL-1 +OSM,IL-1+OSM + retinoic acid, IL-1 +OSM + IL-4, IL-1 +OSM +TGFβ. The medium was har-vested at 24 hours and the levels of TIMP-1 protein measured by ELISA. OSM, retinoicacid, IL-4, and TGF-β all increased levels of TIMP-1, and some further increase in TIMP-1 levels were seen when these agents were combined. Results from a representative exper-iment are shown.

126 ANNALS NEW YORK ACADEMY OF SCIENCES

two of these agents, IL-4 and TGF-β, on reversing the release of collagen fragmentsfrom bovine nasal cartilage to determine whether the upregulation of TIMP-1 wasable to block the action of the collagenases and the subsequent release of collagen.

Control of Cartilage Collagen Breakdown by Control of TIMP-1 and MMP-1

Bovine nasal cartilage was cultured as described in Materials and Methods.IL-1+OSM released >90% collagen by day 14 (FIG. 5). This release was inhibited ina dose-dependent way by increasing concentrations of IL-4 from 2 ng/ml to 50 ng/ml(FIG. 5). Total inhibition of collagen release was seen at 50 ng/ml IL-4. When the lev-els of collagenolytic activity were measured, low collagen release was accompaniedby low collagenolytic activity. Little effect was seen on the levels of TIMPs (data notshown). Collagen release was also inhibited in a dose-dependent way by increasingconcentrations of TGF-β from 1 ng/ml to 50 ng/ml. TGF-β at >10 ng/ml completely

FIGURE 5. The effect of IL-4 on the release of collagen fragments from bovine nasalcartilage treated with IL-1 +OSM. Bovine nasal cartilage was cultured in quadruplicate in600 µL of medium alone, medium + 1 ng/ml IL-1 + 10 ng/ml OSM, medium + IL-1 +OSM+ IL-4 at 2, 10, and 50 ng/ml. Medium was removed on day 7 and replaced and removed atday 14, and the remaining cartilage digested with papain. The levels of OH-proline as ameasure of collagen release were measured in day-7 and -14 medium and the papain di-gests. The % of the total collagen released with each treatment is shown. IL-4 significantlyinhibited the release of collagen by IL-1 +OSM. Results are expressed as mean ± SD.*** = p < 0.001.

127CAWSTON et al.: MMPS & TIMPS IN CARTILAGE TURNOVER

blocked the release of cartilage collagen fragments (FIG. 6) and collagenolytic activ-ity in the culture medium. Little effect was observed on TIMP activity (data notshown). These studies indicate that upregulation of collagenases is required with sub-sequent activation before collagen destruction can occur. Reducing the total activityof the collagenases is sufficient to block cartilage collagen resorption.

FIGURE 6. The effect of TGF-β on the release of collagen fragments from bovine na-sal cartilage treated with IL-1 +OSM. Bovine nasal cartilage was cultured in quadruplicatein 600 µL of medium alone, medium + IL-1 +OSM, and medium + IL-1 +OSM +TGF-β at 1,2.5, 10, 25, and 50 ng/ml. Medium was removed on day 7 and replaced and removed at day14 and the remaining cartilage digested with papain. The levels of OH-proline as a measureof collagen release were measured in day-7 and -14 medium and the papain digests. The %of the total collagen released with each treatment is shown in day-14 medium samples.TGF-β significantly inhibited the release of collagen induced by IL-1 +OSM at concentra-tions greater than 2.5 ng/ml. Results are expressed as mean ± SD. *** = p < 0.001.

128 ANNALS NEW YORK ACADEMY OF SCIENCES

DISCUSSION

Previous studies investigating the release of proteoglycan in response to IL-1alone showed variable effects19 and only 30–40% of these samples responded. Inthis study not only did the inclusion of OSM with IL-1 initiate a more rapid release,but also 98% of the samples tested to date responded by releasing proteoglycan. Therelease of collagen, which is rarely released in response to IL-1 alone, was less dra-matic. Only 50% of the samples responded, and in very few did the level of releaseexceed 20% of the total collagen. Although this effect is greater than with IL-1, alonethe reasons for the variable response are not known. Certainly the samples of humancartilage obtained at joint operations are from an elderly population and the numberof cells in these cartilage samples are relatively low. Other growth factors could beneeded to elicit the responses which are not present in some samples of human car-tilage. Alternatively, inhibitory molecules, which block the response, could bepresent in unresponsive samples. Cartilage is not the only human tissue to respondto IL-1+OSM, as human tendon also releases collagen fragments in response to thiscombination of cytokines.20

Analysis of the proteoglycan fragments from patients’ cartilage in vivo has shownthat the proteoglycan is specifically cleaved between the G1 and G2 domains of hu-man aggrecan to reveal a new N-terminus that has the sequence ARGSV.21 Thiscleavage is effected by an enzyme, putatively named aggrecanase, but it is not yetcharacterized. We used antibodies that specifically recognize this new epitope gen-erated by aggrecan cleavage and showed that an enzyme present on chondrocytemembranes specifically cleaved aggrecan at this point. IL-1+ OSM treatment mark-edly upregulated this activity.

This study also shows that to degrade collagen the collagenases need to be upreg-ulated. Although TIMPs are upregulated early in the response of cartilage to growthfactors, this effect is not sustained. In contrast, the upregulation of the collagenasesis sustained; and once these enzymes are activated, they exceed the local concentra-tion of TIMPs, and collagen destruction then proceeds. IL-4 and TGF-β are able todownregulate the collagenases, which could explain the protection of cartilage col-lagen seen with these growth factors and cytokines in this study.

ACKNOWLEDGMENTS

We thank the Arthritis Research Campaign for their support of this work and Chi-roscience Ltd., Cambridge for supporting the work on aggrecanase.

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