Plasminogen activator inhibitor in human articular cartilage chondrocyte culture

J. Biochem. 104, 960-967 (1988)

Detection and Partial Characterization of a Specific PlasminogenActivator Inhibitor in Human Chondrocyte Cultures1

Harumoto Yamada,*2 Ross W. Stephens,*3 Tomoyuki Nakagawa,** and David McNicol*

'Department of Medicine and Clinical Science, The John Curtin School of Medical Research, Australian NationalUniversity, Canberra, Australia 2605; and "Department of Orthopaedic Surgery, Keio University, School ofMedicine, Shinjuku-ku, Tokyo 160

Received for publication, June 28, 1988

Serum-free culture medium collected from primary monolayer cultures of human articularchondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Althoughchondrocyte culture medium contained a small amount of endothelial-type plasminogenactivator inhibitor which could be demonstrated by reverse fibrin autography, most of theurokinase inhibitory activity of chondrocyte culture medium was shown to be due to adifferent molecule from endothelial-type inhibitor, since it did not react with a specificantibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culturemedium was partially purified by concanavalin A-Sepharose affinity chromatography. Thepartially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mrurokinase, but no obvious inhibition was detected against tissue-type plasminogenactivator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 onsodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodiumdodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor wasaccompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complexbetween them. Inhibition and complex formation required the active site of urokinase. Thepartially purified inhibitor was thought to be immunologically different from the knownclasses of plasminogen activator inhibitors, including endothelial-type inhibitor, macro-phage/monocyte inhibitor, and protease nexin, since it did not react with specific anti-bodies to these inhibitors.

Recent research on extracellular proteolytic reactions hasrevealed that plasminogen activator (PA) could contributeto tissue degradation and remodeling in many importantprocesses. PA may play significant roles through plasmingenerated from plasminogen in tumor invasion and metas-tasis, mammary gland involution, embryo implantation,and inflammation (see Refs. 1 and 2 for reviews). It has alsobeen shown that PA may take part in the extracellularmatrix degradation of articular cartilage under both physi-ological and pathological conditions (3-6).

PA activity is usually regulated by two different mecha-nisms after its secretion; secretion of PA as a proenzymeform and its activation by plasmin (7, 8), and regulation byspecific inhibitors (9). Especially in cartilage matrix degra-dation, the regulation of PA activity by inhibitors is asubject of some interest, since cartilage has been shown tocontain various types of proteinase inhibitors (10-13).

1 This work was supported financially by the Australian OrthopaedicAssociation Research Foundation and Royal Australian College ofSurgeons Research Foundation. H. Yamada was the recipient of aFellowship from Keio-Gijuku Fukuzawa Foundation during his stay inAustralia.Present addresses: 2 Department of Orthopaedic Surgery, KeioUniversity, School of Medicine, Shinjuku-ku, Tokyo 160. 3 Depart-ment of Virology, University of Helsinki, Helsinki, Finland 00290.Abbreviations: PA, plasminogen activator; PA1, plasminogen ac-tivator inhibitor; SDS, sodium dodecyl sulfate; PAGE, poly-acrylamide gel electrophoresis.

These cartilage inhibitors have been reported to regulatecartilage matrix degradation in arthritic disease and also tobe one of the factors which give cartilage tissue specialresistance against tumor and vascular invasions (14-16).To date three different types of PA inhibitors (PAI) havebeen reported to exist in the human body; endothelial-typePA inhibitor (PAI-1) (17, 18), placental or macrophage/monocyte- derived PA inhibitor (PAI -2) (29, 20), andprotease nexin (PN) (21, 22). These inhibitors could inhibitthe locally secreted or cell-bound PA activity and controlthe extracellular matrix degradation by the PA-plasminenzyme cascade system. The present paper describes thedetection, partial purification and characterization of aspecific PA inhibitor in human chondrocyte cultures.

MATERIALS AND METHODS

Cell Isolation and Culture—Human chondrocytes wereisolated from articular cartilage obtained at replacementsurgery or autopsy. Macroscopically normal areas wereselected for the source of research material. Chondrocyteswere isolated by sequential digestion with trypsin (bovine,type III, Sigma Chemical Co., St. Louis, Mo.) at 2 mg/ml inDulbecco's modified Eagle's medium (DMEM, GIBCO,Grand Island, N.Y.) at 37°C for 1 h, followed by 12-18 h at37°C with bacterial collagenase (CLSIV, Cooper Bio-medical, Malvern) at 2 mg/ml in DMEM containing 10%fetal calf serum. The cells were inoculated into monolayer

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Plasminogen Activator Inhibitor in Chondrocyte Culture 961

culture in DMEM containing 10% fetal calf serum, 50 //g/4 ml of ascorbic acid, 2 mM glutamate, 100 u/ml of penicil-

lin, and 100 //g/ml of streptomycin at a cell density of 5 X105 cells/cm2 in 17 mm wells (Lindbro multiwell plates,Flow Labs, McLean, Va.). On the third day the cell layerwas washed three times with serum-free DMEM containing0.3% gelatin to remove all traces of serum. Then the culturewas maintained with serum-free DMEM containing 0.3%

V bovine serum albumin (essentially fatty acid-free, SigmaChemical Co.), 50 // g/ml of ascorbic acid, 2 mM glutamate,5 //g/ml of human transferrin, 100 u/ml of penicillin, and100 //g/ml of streptomycin. The serum-free culturemedium was subsequently changed every day and sampleswere kept at — 70°C after adding NaN3 to a final cone, of

x 0.02% and Tween 20 (Sigma Chemical Co.) to 0.01% for%~ later assay and purification. Chondrocytes which were

obtained by this high cell density culture method showedround and polygonal morphology during the early stage ofculture. To characterize cultured chondrocytes, the mono-layer culture was labeled with 35S-inorganic sulfate(Amersham Australia, Sydney). After labeling, the culture

k medium and cell layer were separately analyzed for newly*" synthesized radio-sulfate labeled macromolecules (proteo-

glycans) after treatment with 4 M guanidine hydrochloridesolution. The extracts were fractionated on a Sepharose 4B(Pharmacia Australia, North Ryde, N.S.W.) column underassociative conditions. The radioactivity remaining afterdialysis showed that 80% of radio-sulfated macromolecules

\r were secreted into medium, and 20% were localized in thecell layer. Over 68% of radioactivity extracted from the celllayer was excluded at the void volume on Sepharose 4B gelchromatography (data not shown). These results suggestedthat the cultured chondrocytes had the ability to synthesizethe aggregate form of proteoglycans and therefore retainedsome of their chondrocyte features at least in the earlyculture stage.

Determination of Inhibitor Activity—PA inhibitor activ-*- ity was assayed by measurement of the residual urokinase

activity after preincubation with samples using the two-step colorimetric assay described by Coleman and Green(23). A 20//I aliquot of each samples was preincubated

^ with 20//I of urokinase (human urinary urokinase, 0.2Ploug units (PU)/ml; high-Mr urokinase: 80,000 PU/mg

L - protein, low-Afr form less than 2%, low-Mr urokinase:^ 160,000 PU/mg protein, high-Mr form less than 2%,

American Diagnostica, Sydney, N.S.W.) in 50 mM glycine,pH 7.8, containing 0.1% Triton X-100, 0.1% gelatin, and 5mM 6-aminocaproic acid at 23°C for 90 min. Then 40 //I ofaffinity-purified human plasminogen (24) (50 //g/ml in theglycine buffer) was added and the mixture was incubated

I/ for 45 min at 37°C. The plasmin generated was assayed bythe addition of thioesterase substrate (Z-lysine-thiobenzylester, Peninsula Laboratories, San Carlos, Calif.) and 1 mlof color agent (23). The mixture was further incubated for30 min at 37°C, and the absorbance was read at 412 nm.

-* One unit of urokinase inhibitor was defined as the amountof inhibitor sufficient to give 50% inhibition of 4 mPU of

y high-Mr urokinase in the two-step colorimetric assay.Plasmin (human, Sigma Chemical Co.) and trypsin (bovinepancreas, type III, Sigma Chemical Co.) inhibition wasassayed using thioesterase substrate as in the second step ofthe urokinase inhibition assay. Thrombin (bovine, SigmaChemical Co.) inhibition was assayed using Spectrozyme

TH (American Diagnostica), a chromogenic substrate foramidolytic activity of thrombin.

Partial Purification of the Inhibitor from ChondrocyteCulture Medium—Chondrocyte culture medium (80 ml)was charged onto a concanavalin A-Sepharose (SigmaChemical Co.) column (0.8x3 cm) equilibrated with 0.01M sodium phosphate buffer, pH7.4, containing 0.02%NaN3 and 0.01% Tween 20 at a flow rate of 4 ml/h at 4°C.The column was washed with 10 column volume of thephosphate buffer containing 0.15 M NaCl at a flow rate of 4ml/h, and then with the phosphate buffer containing 0.5 MNaCl, to remove the non-specifically bound proteins. Thenthe proteins were eluted from the column with a 0-0.2 Mgradient of or-methyl-D-mannoside (Sigma Chemical Co.)in the phosphate buffer containing 0.5 M NaCl at a flow rateof 1.5 ml/h at 4°C. Fractions were desalted using a PD-10column (Pharmacia Australia) equilibrated with the phos-phate buffer. The desalted fractions were measured forabsorbance at 280 nm to estimate protein concentration,and then assayed for urokinase inhibitory activity. Theinhibitory active fractions obtained were used as affinity-purified inhibitor for further experiments.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electro-phoresis (SDS-PAGE) Followed by Fibrin Overlay andReverse Fibrin Autography—The inhibitory effects of thesamples against PAs were characterized using SDS-PAGEand a zymographic technique according to the methoddescribed by Granelli-Piperno and Reich (25). Each samplewas preincubated with PA for 90 min at 23°C, and then themixture was subjected to SDS-PAGE (11% gel). After theelectrophoretic run, the polyacrylamide gel was washed in2.5% Triton X-100 solution for 90 min at 23°C to renaturethe enzymes. Then the polyacrylamide gel was overlayedonto the fibrin gel which was prepared from 2.7 mg/ml offibrinogen (sheep, type X, fraction I, Sigma Chemical Co.),1.1% agarose (Sea Plaque, FMC Corp., Rockland, Maine),15 ng/ml of bovine thrombin (Sigma Chemical Co.), and 36//g/ml of affinity-purified human plasminogen. PA in thepolyacrylamide gel activated plasminogen in the fibrin gel,and plasmin formed was detected by the lysis bandsproduced in the fibrin gel.

For the detection of SDS-stable PA inhibitory activity inchondrocyte culture medium, reverse fibrin autographywas performed according to the method of Loskutoff et al.(26). Chondrocyte culture medium was preincubated withSDS-containing sample buffer (SDS final concentration 2%)at 37°C for 30 min. After electrophoresis and washing with2.5% Triton X-100 solution, the polyacrylamide gel waslaid over the fibrin gel containing 40 mPU/ml of high-Mr

urokinase and 36 //g/ml of plasminogen. This fibrin gel waskept at 37°C for several hours to develop the spontaneouslysis with urokinase-activated plasminogen.

Stability of the Inhibitor—To study the stability of theinhibitor towards a denaturing agent, affinity-purifiedinhibitor at various concentrations was incubated with 0.2%SDS for 30 min at 37'C. The SDS-treated inhibitor wasrenatured by mixed micelle formation with 2% TritonX-100 for 30 min at 23°C, and then the residual inhibitoractivity was assayed using the two-step colorimetric assay.Control samples were treated with distilled water insteadof SDS. The acid stability of the inhibitor was tested at pH2.7 for 30 min at 37°C, by careful addition of cone. HC1solution.

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Radial Diffusion Assay of PA Inhibitor—This was car-ried out using the fibrin gel with the same contents as usedin the fibrin overlay. High-Mr urokinase (20 mPU) or tissuetype PA (two-chain t-PA, from human melanoma cell line,single chain form less than 2%, American Diagnostica) waspreincubated with various amounts of affinity-purifiedinhibitor for 90 min at 23°C, with or without prior SDStreatment of the inhibitor, and then added to the wells cut*in the fibrin gel. The development of the lysis zone causedby the residual PA activity was observed after 20 h ofincubation at 37°C. The fibrin gel with the omission ofplasminogen was used for the detection of plasmin inhibi-tory activity of the inhibitor preparation.

Treatments with Specific Antibodies to Known Classes ofPA Inhibitors—Immuno-crossreactivities of the inhibi-tors) in chondrocyte culture medium with three differenttypes of PA inhibitors were studied using specific antibodiesand protein A-Sepharose CL-4B (Pharmacia Australia)columns. Rabbit immunoglobulin to PAI-1 was a generousgift from Dr. E.D. Sprengers, Gaubius Institute, Leiden.Rabbit antiserum to PAI-2 was from Dr. E. Kruithof,Lausanne, Switzerland. Rabbit immunoglobulin to PN waskindly provided by Dr. J. Baker, University of Kansas,Lawrence, KS. Chondrocyte culture medium (300^1) wasincubated with 10//I of immunoglobulin or antiserum for90 min at 23°C. The mixtures were then applied to a proteinA-Sepharose column (200//I) equilibrated with the phos-phate buffer. The pass-through fractions were collected andassayed for residual urokinase inhibitory activities usingthe two-step colorimetric assay. The fractions were alsoapplied to SDS-PAGE followed by fibrin overlay andreverse fibrin autography. Immuno-crossreactivities of theaffinity-purified inhibitor with these known PA inhibitorswere also studied using the same immunoadsorptionmethod.

RESULTS

Inhibition of Urokinase Activity by Chondrocyte CultureMedium—Serum-free chondrocyte culture medium inhib-ited dose-dependently the two-step colorimetric assay ofurokinase activity as shown in Fig. 1. Day 3 chondrocyteculture medium (20 ^1) produced 95% inhibition against 4mPU of high-Mr urokinase in this assay system. Culturedchondrocytes continued to produce a significant level ofurokinase inhibitory activity in the medium for at least 5 d.Further observation revealed that chondrocyte culturemedium from the 14th day still had 47% inhibitory activityagainst 4 mPU of high-Mr urokinase (data not shown). Thisurokinase inhibitory activity of the medium was sup-

pressed by the addition of the protein synthesis inhibitor,cycloheximide, at a concentration of 3 /ig/ml. Cell lysatesof chondrocytes also had urokinase inhibitory activity,which gradually decreased during the culture period. Theinhibitory activity of the cell lysates was also suppressed bythe addition of cycloheximide (Fig. 2). The inhibitoryactivity of chondrocyte culture medium against plasminwas studied using the plasmin assay reagents (23) in theabsence of plasminogen: 20fi\ of the day 3 mediumproduced only 7% inhibition of 2 ng of plasmin, the equiva-lent amount of plasmin generated from 2 /* g of plasminogenby 4 mPU of high-Mr urokinase in this assay system. Theseresults suggested that the inhibition was mainly at the level

2 100

1 2 3 4 SDays after changing the medium (days)

to serum tree DMEM

Fig. 2. Secretion of urokinase inhibitory activity by chon-drocyte culture. Chondrocytes were inoculated in DMEM containing10% fetal calf serum at a cell density of 5 x 105 cells/cm2. From thethird day after inoculation, the cultures were harvested with serum-free DMEM containing 0.3% bovine serum albumin without cyclohex-imide (O) or with 3 pi g/ml of cycloheximide ( • ) . Serum- free culturemedium (0.25 ml/cm2) was replaced every 24 h. Cell lysates of theculture without cycloheximide (A) and with cycloheximide (*) wereextracted with 0.5% Triton X-100, 50 mM glycine pH 7.8 (0.25 ml/cm2) at 4'C for 1 h.

I<•* 0 — 92K

— 67K

— 43K

— 30K

-4.

2" 2 ' 2"Dilution factor

Fig. 1. Titration of urokinasewith chondrocyte culturemedium. Day 3 chondrocyteculture medium was dilutedwith non-conditioned mediumas indicated. Then 20u^ ofdiluted chondrocyte culturemedium was incubated withhigh-Mr urokinase (4 mPU in20 ̂ 1) at 23*C for 90 min. Theresidual urokinase activity wasassayed by the two-step color-imetric assay.

Fig. 3. Fibrin overlay of urokinase with chondrocyte culturemedium. A mixture of high- and low-Afr urokinase (4 mPU, lanes 2-6) was incubated with 20//I (lane 3), 40^1 (lane 4), 60//I (lane 5),and 80 n 1 (lane 6) of chondrocyte culture medium at 23'C for 90 min.The mixtures were electrophoresed and then overlayed on fibrin gelsas described under "MATERIALS AND METHODS." Lane 1, 80y\of chondrocyte culture medium without any additions, lane 2, 4 mPUof urokinase with 80^1 of non-conditioned medium. The Mr ofmarker proteins were estimated using human plasminogen (92K,92,000), bovine serum albumin (67K, 67,000), egg white ovalbumin(43K, 43,000), and carbonic anhydrase (30K, 30,000) as standards.

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— 92K

— 67K

— 43K

— 30K

5 6 7 8 9

Fig. 4. Effects of antibodies to three types of PA inhibitors.Chondrocyte culture medium was incubated with antibodies to PAinhibitors, then applied to a protein A-Sepharose column. Thepass-through fractions were assayed for lysis-resistant band forma-tion in reverse fibrin autography (lanes 1-4), and for high-Mr complexformation in fibrin overlay (lanes 5-9). Lanes 1, 6: treated withnon-immunized immunoglobulin; lanes 2, 7: treated with antibody toPAI-1; Lanes 3,8: treated with antibody to PAI-2; Lanes 4,9: treatedwith antibody to PN; Lane 5: 4 mPU of high-Mr urokinase withoutany addition.

7.0

6.9

;

0.3

0.2

0.1

CCM 0.15M NaCI 0.5MNaCI 0-0.2MJ/ ^ ^ yh MM

100 =

80 90 100Fraction volume (ml)

110

Fig. 5. Profile of chondrocyte culture medium on concanavalinA-Sepharose affinity chromatography. Serum-free culturemedium (80 ml) from human chondrocyte culture was applied to aconcanavalin A-Sepharose column (0.8 x 3 cm) equilibrated with 0.01M sodium phosphate buffer pH 7.4 containing 0.02% NaN3 and 0.01%Tween 20. The column was eluted stepwise with the same buffercontaining 0.15 M NaCI, 0.5 M NaCI, and then a 0-0.2 M lineargradient of ar-methyl-D-mannoside. Fractions were measured forabsorbance at 280 nm (for protein determination) ( • ) , and assayedfor inhibitory activity against high-Mr urokinase using the two-stepcolorimetric assay (o) . CCM, chondrocyte culture medium; MM,a - methyl • D- mannoside.

— 92K

— 67K

— 43K

— 30K Fig. 6. SDS-PAGE of partially purified in-hibitor. Affinity-purified inhibitor (5//g) waselectrophoresed on 11% SDS polyacrylamide gel.Proteins in the gel were stained using Coomassieblue.

o 0.01 0.1 1 10 100 1000

Inhibitor concentration (yg/ml)

Fig. 7. Titration of urokinase with partially purified in-hibitor. Various amounts of affinity-purified inhibitor were assayedfor inhibitory activity against 4 mPU of high-Mr urokinase, withoutany treatment ( • ) , or with 0.2% SDS treatment prior to thisexperiment (o) . After incubation for 90 min at 23'C, the residualenzyme activities of the mixtures were assayed using the two-stepcolorimetric assay.

100

20 40 60Incubation time (min)

80

Fig. 8. Inhibition of low- and high-Mr urokinase with par-tially purified inhibitor. Low- ( • ) and high- ( • ) MT urokinase (4mPU) were incubated with 2/*g of affinity-purified inhibitor forvarious times at 23'C. Then the residual urokinase activity wasassayed using the two-step colorimetric assay.

TABLE I. Specificity of affinity-purified inhibitor for serineproteinases. Affinity-purified inhibitor (80 ng) was incubated witheach amount of enzyme for 90 min at 23'C. Then the residual enzymeactivities were assayed as described under "MATERIALS ANDMETHODS." Sodium phosphate buffer, pH 7.4, containing 0.01%Tween 20 was used for each control experiment instead of theinhibitor preparation.

Enzyme

UrokinasePlasminTrypsinThrombin

Enzymeamount

4mPU2ng

25 ng6.4 ng

Residual enzyme activity(absorbance at 412 nm)

Control

0.730.690.830.43

Inhibitor-treated0.050.690.810.45

%inhibition

93010

of plasminogen activation and not simple inhibition ofplasmin.

Characterization of Urokinase Inhibitory Activity inChondrocyte Culture Medium Subjected to SDS-PAGE—The lysis bands of urokinase on SDS-PAGE followed byconventional fibrin overlay were quenched dose-depen-dently by the addition of chondrocyte culture medium.High-Mr urokinase seemed to be more susceptible than

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Fig. 9. Radial diffusion assay of partially purified inhibitor.For this assay, 20 mPU of high-Mr urokinase (rows, 1, 2), 20 mPU oft-PA (rows 3, 4), or 500 ng of human plasmin (row 5) was incubatedwith no addition (lane A), or 0.3 n% (lane B), 0.6//g (lane C), or 1.2tig (lane D) of affinity-purified inhibitor for 90min at 23'C. Themixtures were charged into the wells cut in the fibrin gels containingplasminogen (rows 1-4) or without plasminogen (row 5), and thenkept at 37°C for 20 h. For lanes 2 and 4, the inhibitor was treated with0.2% SDS followed by 2% Triton X-100, prior to this experiment.

— 92K

— 67K

_ 4 3 K

TABLE II. Stability of affinity-purified inhibitor. Affinity-purified inhibitor (80 ng) (in 0.01 M sodium phosphate buffer, pH 7.4containing 0.01% Tween 20) was acidified to pH 2.7 by addition ofHC1 solution and kept at 37'C for 30 min. Then the pH was raised topH 7.4 by addition of NaOH solution. The same amounts of HC1 andNaOH solutions were added to the non-treated sample at the sametime and kept for 30 min at 37'C. Residual urokinase inhibitoryactivity of the mixture was assayed using the two-step colorimetricassay, after dialysis against the phosphate buffer. The same phos-phate buffer was used for both control experiments instead of theinhibitor preparation.

Treatment Residual urokinase activity(absorbance at 412 nm)

% inhibition

AcidControlTreatedNon-treated

HeatControlTreatedNon-treated

0.980.700.29

0.590.580.09

—2970

—2

85

TABLE III. Effects of antibodies to known PA inhibitors onaffinity-purified inhibitor. Affinity-purified inhibitor (0.5 us) w a streated with antibodies to three types of PA inhibitors, then chargedto protein A-Sepharose column. The pass through fractions wereassayed for urokinase inhibitory activity using the two-step color-imetric assay. Affinity-purified inhibitor was replaced by phosphatebuffer for the control experiment.

Treatment withantibody toControlNon- immunizedPAI-1PAI-2PN

Residual enzyme activity(absorbance at 412 nm)

0.840.450.400.430.39

—46524853

— 30K

Fig. 10. Effects of partially purified inhibitor on PAs (fibrinoverlay). For this experiment, 4 mPU of high-Mr urokinase (lanes 1 -5) or low- Mr urokinase (lanes 6-10) was incubated with 0.25 fig (lanes2, 7), 1.0 jig (lanes 3, 8), or 4.0//g (lanes 4, 9) of affinity-purifiedinhibitor for 90 min at 23'C. Then the mixtures were electrophoresedon ll%SDS-polyacrylamidegels. After soaking in 2.5% TritonX-lOOsolution 90 min at 23'C, the gels were overlayed onto the fibrin gelscontaining plasminogen. The development of lysis bands was per-formed in a humidified box at 37'C for 10 h. Lanes 1,6 were enzymeswithout any addition. Lanes 5, 10 were the same as lanes 4, 9,respectively, but plus 40 mM p-aminobenzamidine.

low-Mr urokinase (Fig. 3, lanes 3-6). Concurrent with thequenching of the original urokinase activity, weak lysisbands appeared at Mr of 74,000 and 94,000. These newhigh-Mr lysis bands were also dose-dependently increasedin intensity in proportion to the amount of the mediumadded (Fig. 3, lanes 3-6). These observations suggestedthat chondrocyte culture medium contained inhibitor(s)which had affinity to urokinase and formed high-Mr SDS-stable complexes with urokinase.

Effects of Antibodies to Known Classes of PA Inhibitorson Urokinase Inhibitory Activity of Chondrocyte CultureMedium—On SDS-PAGE of chondrocyte culture medium

followed by reverse fibrin autography, a weak lysis-resistant band was occasionally observed with apparent Mr

of 52,000. To verify that this lysis-resistant band wascaused by the inhibition of urokinase-mediated plas-minogen activation and not by simple inhibition of plasmin,urokinase was replaced by streptokinase with a similaractivity in the fibrin gel. No obvious lysis-resistant bandwas observed on this fibrin gel (figure not shown). Thelysis-resistant band of chondrocyte culture medium inreverse fibrin autography with Mr of 52,000 was complete-ly abolished by immunoadsorption treatment with antibodyto PAI-1 (Fig. 4, lane 2). However, this treatment had nosignificant effect on the urokinase inhibitory activity of themedium observed in the two-step colorimetric assay (datanot shown), or on the Mr 94,000 complex formed withhigh-Mr urokinase as seen by fibrin overlay (Fig. 4, lane 7).The treatments with antibody to PAI-2, and PN had nosignificant effect on the urokinase inhibitory activity of themedium as seen in the two-step colorimetric assay (data notshown), the Mr 52,000 lysis-resistant band in reverse fibrinautography (Fig. 4, lanes 3 and 4), or the Mr 94,000complex formation in fibrin overlay (Fig. 4, lanes 8 and 9).These observations revealed that chondrocyte culturemedium contained at least two separate urokinase in-hibitors, PAI-1 as the minor inhibitor, and an inhibitordifferent from known classes of PA inhibitors as the majorinhibitor.

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Purification of the Inhibitor from Chondrocyte CultureMedium—Serum-tree chondrocyte culture medium (80ml) containing urokinase inhibitor was applied to a con-canavalin A-Sepharose column equilibrated with the phos-phate buffer containing 0.15 M NaCl. Urokinase inhibitorwas eluted from the column in the early stage of a 0-0.2 Mar-methyl-D-mannoside gradient with a small but detect-able protein peak (Fig. 5). Most of the pass-through proteinwas bovine serum albumin which had been added to the cellculture medium. Ninety-nine percent of the protein in themedium passed through the column unbound, and 31% ofthe urokinase inhibitory activity was recovered in thefractions eluted with <r-methyl-D-mannoside with 199-foldpurification. Material eluted from the concanavalin A-Sepharose column with a-methyl-D-mannoside contained amajor protein band with apparent Mr 43,000 on SDS-PAGEand in addition several minor contaminating bands rep-resenting 5-10% of the total Coomassie-stained proteinoptical density (Fig. 6). The contaminating proteins variedwith each batch of conditioned medium but usually account-ed for less than 10% of the total protein. Material from theconcanavalin A-Sepharose column was concentrated andthen fractionated on a Sephacryl S-200 (Pharmacia Aus-tralia) column (1x56 cm) to remove the contaminants.Urokinase inhibitory activity was eluted from the columnas a single peak with Mr 45,000 (figure not shown), howeverthis procedure resulted in a great loss of inhibitor activity.So material from affinity chromatography was used aspartially purified inhibitor in this experiment.

Specificity of the Partially Purified Inhibitor in theColorimetric Assay—The affinity-purified inhibitor inhib-ited high-Mr urokinase dose-dependently in the two-stepcolorimetric assay. Treatment of the affinity-purified in-hibitor with a denaturing agent, 0.2% SDS, prior to theassay produced a significant loss in urokinase inhibitoryactivity (Fig. 7). Inhibition of the same amount of high- andlow-Mr urokinase with the affinity-purified inhibitor isshown in Fig. 8. While the inhibition of high-Mr urokinasewas very fast, low-Mr urokinase was inhibited very slowly.

Affinity-purified inhibitor showed no obvious inhibitionagainst the lysine thioesterase activity of plasmin ortrypsin. No inhibition of the amidolytic activity of throm-bin with the inhibitor could be detected using a chromogenicsubstrate (Table I).

Effects of the Partially Purified Inhibitor on Fibrinolysis—Affinity-purified inhibitor inhibited fibrinolysis by uro-kinase strongly in the radial diffusion assay (Fig. 9, row 1).Affinity-purified inhibitor irreversibly lost its urokinaseinhibitory activity after SDS treatment in this assaysystem (Fig. 9, row 2). Against t-PA, no obvious inhibitionwas detected (Fig. 9, row 3). No enhancement of t-PAinhibitory activity was observed after SDS treatment (Fig.9, row 4). No inhibition against 20 ng of plasmin wasdetected in this assay system, even with the highestconcentration of inhibitor preparation (Fig. 9, row 5). Thisamount of plasmin gave the same lysis zone as theurokinase-activated plasminogen above.

Inhibition of PAs and Complex Formation with PartiallyPurified Inhibitor as Shown by SDS-PAGE Followed byFibrin Overlay—High-Mr urokinase and low-Mr urokinase(4 mPU) were each preincubated with various amounts ofaffinity-purified inhibitor for 90 min at 23°C. Then theresidual enzyme activities were visualized as lysis bands on

fibrin overlay. Control high-Mr urokinase showed a clearlysis band at the position of Mr 54,000 after a 10 h incuba-tion (Fig. 10, lane 1). By the addition of increasing amountsof inhibitor, the lysis bands of high-Mr urokinase werequenched dose-dependently (Fig. 10, lanes 2-4). At thesame time as quenching of Mr 54,000 lysis bands, new clearlysis bands appeared with Mr 94,000 (Fig. 10, lanes 2-4).The quenching of low-Mr urokinase lysis bands with MT

33,000 was significantly weaker compared with the quench-ing of the lysis bands of the same amount of high-Mr

urokinase (Fig. 10, lanes 7-9).The formation of new lysis bands related to low-Mr

urokinase was not clear after 10 h incubation, but a newlysis band appeared cleary with Mr 74,000 after 20 h ofincubation (not shown). To verify that the inhibition of PAenzyme activity by the chondrocyte inhibitor involves theactive site of the enzyme, each PA was preincubated withinhibitor in the presence of 40 mM p-aminobenzamidine. Inthe presence of 40 mM p-aminobenzamidine, the quench-ing of the lysis bands with Mr 54,000 (high-Mr urokinase)and 33,000 (low-Mr urokinase) was completely inhibited(Fig. 10, lanes 5 and 10). The same treatment also inhibitedthe formation of the new lysis bands with Mr 94,000 (withhigh-Afr urokinase, Fig. 10, lane 5) and 74,000 (withlow-Mr urokinase, not shown) completely.

Other Characteristics of the Partially Purified Inhibitor—Affinity-purified inhibitor lost about 60% of its urokinaseinhibitory activity after acidification at pH 2.7 at 37°C for30 min, and 98% of the inhibitory activity was destroyedduring a 30 min incubation at 60°C (Tablell). The treat-ments with antibodies to known classes of PA inhibitors,PAI-1, PAI-2, and PN, had no significant effect on theurokinase inhibitory activity of the affinity-purified in-hibitor as seen in the two-step colorimetric assay (TableIII). Furthermore, these treatments had no effect on the Mr

94,000 complex formation of the affinity-purified inhibitorwith high-Mr urokinase in fibrin overlay (not shown).

DISCUSSION

The present data show that serum-free conditionedmedium from primary human chondrocyte culturecontained a specific inhibitor of PA. The inhibition ofurokinase enzyme activity with crude chondrocyte culturemedium was initially demonstrated by the two-step color-imetric assay. This coupled assay method strongly suggest-ed the presence of urokinase inhibitor in the culturemedium. The inhibitor in the crude culture medium waspartially purified using concanavalin A-Sepharose affinitychromatography. The partially purified inhibitor wasspecific to PA, since it showed no obvious inhibition againstother serine proteinases including plasmin, trypsin, andthrombin. The specific inhibition of urokinase with par-tially purified inhibitor was confirmed by the followingmethods: 1) two-step colorimetric assay, 2) radial diffusionassay, and 3) SDS-PAGE followed by fibrin overlay.

There are two forms of urokinase; high-Mr. urokinasewith Mr of 53,000, and low-Mr urokinase with Mr of 33,000.Low-Mr urokinase is a form derived from high-Mr uro-kinase by limited proteolytic cleavage, which releases apolypeptide of 157 amino acids (27). These two forms arereported to be enzymatically and immunologically similar(28). Chondrocyte PA inhibitor inhibited high-Mr uro-

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kinase more effectively than low-Mr urokinase in thetwo-step colorimetric assay. The mechanism of the resist-ance of low-Mr urokinase to the inhibition with chon-drocyte PA inhibitor has not been clarified in this study.The low susceptibility of low-Mr urokinase to chondrocytePA inhibitor may be nearly unique, since a similar phenom-enon has only been reported in the case of antithrombin III(29), and not in the case of known classes of specific PAinhibitor.

It has been reported that t-PA requires a stimulator suchas fibrin for the interaction with plasminogen (30). Radialdiffusion assay, in which the interaction of PA with plas-minogen was performed in the presence of fibrin, wassuitable for the determination of the PA inhibition spec-trum, since the two-step colorimetric assay was specific forthe urokinase-type PA, in that t-PA had little or no activityunder the present assay conditions. The result of radialdiffusion assay showed that the partially purified inhibitorhad a high affinity to urokinase, but showed no obviousinhibition against t-PA and plasmin in this assay system.SDS-PAGE followed by fibrin overlay also demonstratedthat the partially purified inhibitor inhibited urokinaseenzyme activity dose-dependently, and formed a newhigh-Mr lysis band. It has been reported that SDS treat-ment induces a conformational change of such an enzyme -inhibitor complex, in which the complex regains enzymeactivity after SDS treatment (31). So it is reasonable tospeculate that the new high-Mr lysis band was the complexof PA and PA inhibitor. By this assay method, it was alsodemonstrated that high-Mr urokinase could be inhibitedmore effectively than low-Mr urokinase. p-Aminobenzami-dine, an active site blocker of serine proteinases (32),prevented the inhibitions and complex formation of PAswith partially purified inhibitor, as shown on fibrin overlay.The interaction of p-aminobenzamidine with enzymes wasreversible after treatment of SDS-polyacrylamide gel inTriton X-100 solution, and enzymes treated with p-aminobenzamidine showed similar lysis bands to the con-trols without any effect of inhibitor. These observationssuggested that the inhibition and complex formations ofPAs with inhibitor required the active sites of the enzymes.

To date, three different types of PA inhibitors have beendemonstrated both in cultures of a variety of human cellsand tissues (for review, see Ref. 9). It was clearly estab-lished that the crude chondrocyte culture medium con-tained a small amount of inhibitor immunologically similarto PAI-1, since a weak lysis-resistant band with Mr 52,000as shown in reverse fibrin autography disappeared afterimmunoadsorption using antibody to PAI-1 and proteinA-Sepharose. Sprengers et al. reported that part of thePAI-1 in endothelial cell culture was secreted as a formwhich could be detected only in SDS-PAGE followed byreverse fibrin autography (33). Denaturation by SDStreatment was thought to activate the latent form of theinhibitor (26). However, the major PA inhibitor in chon-drocyte culture medium was shown to be immunologicallynon-identical to PAI-1, because the treatment with specificantibody to PAI-1 had no significant effect on urokinaseinhibitory activity of chondrocyte culture medium in thetwo-step colorimetric assay, and chondrocyte culturemedium treated with antibody to PAI-1 still retained theability to form a new high-Afr complex with urokinase. Themajor PA inhibitor in the crude chondrocyte culture

medium also had no immunological crossreactivity withPAI-2 or PN, since the specific antibodies to these two PAinhibitors had no significant effects on urokinase inhibitoryactivity of chondrocyte culture medium in the two-stepcolorimetric assay, the lysis-resistant band formation inreverse fibrin autography, or the ability to form a high-Mr

complex in fibrin overlay.The partially purified inhibitor did not inhibit t-PA as

shown in radial diffusion assay. The partially purifiedinhibitor was unstable to acid, heat and SDS treatments.These characteristics make it more unlikely that thepresent PA inhibitor is identical to PAI-1, since PAI-1 hasbeen reported to be a potent inhibitor against t-PA andunusually stable to these denaturing conditions (26).Furthermore, the partially purified inhibitor lackedimmuno-crossreactivity with PAI-1 as shown using thespecific antibody to PAI-1 in the two-step colorimetricassay. On the other hand, these instabilities of partiallypurified inhibitor were compatible with the characteristicsof PAI-2 (34, 35). Wholwend et al. reported that humanmacrophage/monocytes produced two forms of PAI-2 withthe same immunological reactivities to antibody; low-Mr

form without glycosylation, and high-Mr form with glyco-sylation (35). High-Mr form of PAI-2 with Mr ranging from50,000 to 65,000 could be adsorbed on concanavalinA-Sepharose due to its glycosylation. However, the studyusing the specific antibody to PAI-2 revealed that thepartially purified inhibitor was not immunologically identi-cal to PAI-2. The partially purified inhibitor was alsothought to be different from PN, since PN was a potentinhibitor of a wide range of serine proteinases includingplasmin, trypsin, and thrombin, unlike the partially purifiedinhibitor (36). Furthermore, the partially purified inhibitorlacked immuno-crossreactivity with PN, since the specificantibody to PN had no effect on the urokinase inhibitoryactivity of the partially purified inhibitor in the two-stepcolorimetric assay. A urinary urokinase inhibitor wasdescribed which is presently shown to be immunologicallyidentical with plasma protein C inhibitor (37, 38).Immuno-crossreactivity with the urinary urokinase in-hibitor was not studied in this experiment. However, thepartially purified inhibitor seemed to be different from theurinary urokinase inhibitor, since the urinary urokinaseinhibitor is a potent inhibitor against thrombin (37).

In conclusion, human articular cartilage chondrocytesproduced a PA-specific inhibitor with a high affinity tohigh-Mr urokinase, the characteristics of which weredistinguishable from those of known classes of PA in-hibitors. Whether chondrocytes in vivo would also producethe same inhibitor as in culture cannot be inferred fromthese experiments. The exact role of this inhibitor in thecell functions also still remain to be clarified. A betterunderstanding of the protease/protease inhibitor balance inthe extracellular matrix degradation of cartilage, may beprovided by further studies.

We thank Drs. R. Kitchin, G. Stubbs, W. Coyle, J.A. Calder, P.Morris, G. Maclaine, and staff of the ACT Health Authority for theircooperation in providing surgical and autopsy materials. Deep grati-tude is expressed to Dr. S. Izumida, Dr. T. Sakamaki, and ProfessorY. Yabe of Department of Orthopaedic Surgery, Keio University,School of Medicine, for their kind encouragement.

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REFERENCES

1. Dano, K., Andreasen, P.A., GrfSndahl-Hansen, J., Kristensen, P.,Nielsen, L.S., & Skriver, L. (1985) Adv. Cancer Res. 44, 139-266

2. Saksela, 0. (1985) Biochim. Biophys. Acta 823, 35-653. Lack, C.H. (1958) Nature 182, 948-9494. Mochan, E. & Uhl, J. (1984) J. Rheumatol. 11, 123-1285. Elford, P.R., Meats, J.E., Sharrard, R.M., & Russel, R.G.G.

(1985) FEBS Lett. 179, 247-2516. Richardson, H.J., Elford, P.R., Sharrard, R.M., Meats, J.E., &

Russel, R.G.G. (1985) Cell. Immunol. 90, 41-517. Wun, T.Z., Ossowski, L., & Reich, E. (1982) J. Biol. Chem. 257,

• 7262-72688. Sim, P.S., Fayle, D.R.H., Dow, W.F., & Stephens, R.W. (1986)

Eur. J. Biochem. 158, 537-5429. Sprengers, E.D. & Kluft, C. (1987) Blood 69, 381-387

10. Kuettner, K.E., Croxen, R.L., Eisenstein, R., & Sorgente, N.(1974) Experientia 30, 595-597

11. Killacky, J.J., Roughley, P.J., & Mort, J. (1983) Coll. Relat. Res.3, 419-430

12. Dean, D.D. & Woessner, J.F. (1984) Biochem. J. 218, 277-28013. Ghosh, P., Andrews, J.L., Osborne, R.A., & Lesjak, M.S. (1986)

Agents Actions Suppl. 18, 69-8114. Eisenstein, R., Kuettner, K.E., Neapolitan, C, Soble, L.W., &

Sorgente, N. (1975) Am. J. Pathol. 81, 337-34815. Stephens, R.W., Ghosh, P., & Taylor, T.K.F. (1979) Med.

Hypotheses 5, 809-81716. Yamada, H., Nakagawa, T., Stephens, R.W., & Nagai, Y. (1987)

Biomed. Res. 8, 289-30017. Loskutoff, D.J. & Edgington, T.S. (1981) J. Biol. Chem. 256,

4142-414518. Andreasen, P.A., Nielsen, L.S., Kristensen, P., Grfindahl-

Hansen, J., Skriver, L., & Dan^, K. (1986) J. Biol. Chem. 261,7644-7651

19. Golder, J.P. & Stephens, R.W. (1983) Eur. J. Biochem. 136,517-522

20. Wun, T.C. & Reich, E. (1987) J. Biol. Chem. 262, 3646-365321. Baker, J.B., Low, D.A., Simmer, R.L., & Cunningham, D.D.

(1980) Cell 21, 37-4522. Baker, J.B. & Gronke, R.S. (1986) Semn. Thromb. Hemost. 12,

216-22023. Coleman, P.L. & Green, G.D.J. (1981) Ann. N. Y. Acad. Sci. 370,

617-62624. Deutsch, D.G. & Mertz, E.T. (1970) Science 170, 1095-109625. Granelli-Piperno, A. & Reich, E. (1978) J. Exp. Med. 148, 223-

23426. Loskutoff, D.J., Van Mourik, J.A., Erickson, L.A., & Lawrence,

D. (1983) Proc. Natl. Acad. Sci. U.S. 80, 2956-296027. Giinzler, W.A., Steffens, G.J., Otting, F., Kim, S.M.A., Frankus,

E., & FlohS, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363,1155-1165

28. White, W.F., Barlow, G.H., & Mozen, M.M. (1966) Biochemistry5, 2160-2169

29. Murano, G., Aronson, D., Williams, L., & Brown, L. (1980)Blood 55, 430-436

30. Hoylaerts, M., Rijken, D.C., Lijnen, H.R., & Collen, D. (1982) J.Biol. Chem. 257, 2912-2919

31. Levin, E.G. (1983) Proc. Natl. Acad. Sci. U.S. 80, 6804-680832. Coats, E.A. (1973) J. Med. Chem. 16, 1102-110633. Sprengers, E.D., Verheijen, J.H., Hinsbergh, V.W.M., & Emeis,

J.J. (1984) Biochim. Biophys. Acta 801, 163-17034. Saksela, O., Hovi, T., & Vaheri, A. (1985) J. Cell. Phys. 122,

125-13235. Wohlwend, A., Belin, D., & Vassalli, J.D. (1987) J. Exp. Med.

165, 320-33936. Scott, R.W. & Baker, J.B. (1983) J. Biol. Chem. 258, 10439-

1044437. Stump, D.C., Thienpont, M.F & Collen, D. (1986) J. Biol. Chem.

261, 12759-1276638. Heeb, M.J., Espana, F., Geiger, M., Collen, D., Stump, D.C., &

Griffin, J.H. (1987) J. Biol. Chem. 262, 15813-15816

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Plasminogen activator inhibitor in human articular cartilage chondrocyte culture