Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures

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

Detection and Partial Characterization of a Specific Plasminogen Activator 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 National University, Canberra, Australia 2605; and "Department of Orthopaedic Surgery, Keio University, School of Medicine, Shinjuku-ku, Tokyo 160 Received for publication, June 28, 1988

Recent research on extracellular proteolytic reactions has revealed that plasminogen activator (PA) could contribute to tissue degradation and remodeling in many important processes. PA may play significant roles through plasmin generated from plasminogen in tumor invasion and metastasis, mammary gland involution, embryo implantation, and inflammation (see Refs. 1 and 2 for reviews). It has also been shown that PA may take part in the extracellular matrix degradation of articular cartilage under both physiological and pathological conditions (3-6). PA activity is usually regulated by two different mechanisms after its secretion; secretion of PA as a proenzyme form and its activation by plasmin (7, 8), and regulation by specific inhibitors (9). Especially in cartilage matrix degradation, the regulation of PA activity by inhibitors is a subject of some interest, since cartilage has been shown to contain various types of proteinase inhibitors (10-13). 1

This work was supported financially by the Australian Orthopaedic Association Research Foundation and Royal Australian College of Surgeons Research Foundation. H. Yamada was the recipient of a Fellowship from Keio-Gijuku Fukuzawa Foundation during his stay in Australia. Present addresses: 2 Department of Orthopaedic Surgery, Keio University, School of Medicine, Shinjuku-ku, Tokyo 160. 3 Department of Virology, University of Helsinki, Helsinki, Finland 00290. Abbreviations: PA, plasminogen activator; PA1, plasminogen activator inhibitor; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

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These cartilage inhibitors have been reported to regulate cartilage matrix degradation in arthritic disease and also to be one of the factors which give cartilage tissue special resistance against tumor and vascular invasions (14-16). To date three different types of PA inhibitors (PAI) have been reported to exist in the human body; endothelial-type PA inhibitor (PAI-1) (17, 18), placental or macrophage/ monocyte- derived PA inhibitor (PAI -2) (29, 20), and protease nexin (PN) (21, 22). These inhibitors could inhibit the locally secreted or cell-bound PA activity and control the extracellular matrix degradation by the PA-plasmin enzyme cascade system. The present paper describes the detection, partial purification and characterization of a specific PA inhibitor in human chondrocyte cultures. MATERIALS AND METHODS Cell Isolation and Culture—Human chondrocytes were isolated from articular cartilage obtained at replacement surgery or autopsy. Macroscopically normal areas were selected for the source of research material. Chondrocytes were isolated by sequential digestion with trypsin (bovine, type III, Sigma Chemical Co., St. Louis, Mo.) at 2 mg/ml in Dulbecco's modified Eagle's medium (DMEM, GIBCO, Grand Island, N.Y.) at 37°C for 1 h, followed by 12-18 h at 37°C with bacterial collagenase (CLSIV, Cooper Biomedical, Malvern) at 2 mg/ml in DMEM containing 10% fetal calf serum. The cells were inoculated into monolayer J. Biochem.

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

Plasminogen Activator Inhibitor in Chondrocyte Culture

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Vol. 104, No. 6, 1988

TH (American Diagnostica), a chromogenic substrate for amidolytic activity of thrombin. Partial Purification of the Inhibitor from Chondrocyte Culture Medium—Chondrocyte culture medium (80 ml) was charged onto a concanavalin A-Sepharose (Sigma Chemical Co.) column (0.8x3 cm) equilibrated with 0.01 M sodium phosphate buffer, pH7.4, containing 0.02% NaN3 and 0.01% Tween 20 at aflowrate of 4 ml/h at 4°C. The column was washed with 10 column volume of the phosphate buffer containing 0.15 M NaCl at aflowrate of 4 ml/h, and then with the phosphate buffer containing 0.5 M NaCl, to remove the non-specifically bound proteins. Then the proteins were eluted from the column with a 0-0.2 M gradient of or-methyl-D-mannoside (Sigma Chemical Co.) in the phosphate buffer containing 0.5 M NaCl at aflowrate of 1.5 ml/h at 4°C. Fractions were desalted using a PD-10 column (Pharmacia Australia) equilibrated with the phosphate buffer. The desalted fractions were measured for absorbance at 280 nm to estimate protein concentration, and then assayed for urokinase inhibitory activity. The inhibitory active fractions obtained were used as affinitypurified inhibitor for further experiments. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Followed by Fibrin Overlay and Reverse Fibrin Autography—The inhibitory effects of the samples against PAs were characterized using SDS-PAGE and a zymographic technique according to the method described by Granelli-Piperno and Reich (25). Each sample was preincubated with PA for 90 min at 23°C, and then the mixture was subjected to SDS-PAGE (11% gel). After the electrophoretic run, the polyacrylamide gel was washed in 2.5% Triton X-100 solution for 90 min at 23°C to renature the enzymes. Then the polyacrylamide gel was overlayed onto the fibrin gel which was prepared from 2.7 mg/ml of fibrinogen (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 the polyacrylamide gel activated plasminogen in the fibrin gel, and plasmin formed was detected by the lysis bands produced in the fibrin gel. For the detection of SDS-stable PA inhibitory activity in chondrocyte culture medium, reverse fibrin autography was performed according to the method of Loskutoff et al. (26). Chondrocyte culture medium was preincubated with SDS-containing sample buffer (SDSfinalconcentration 2%) at 37°C for 30 min. After electrophoresis and washing with 2.5% Triton X-100 solution, the polyacrylamide gel was laid over the fibrin gel containing 40 mPU/ml of high-Mr urokinase and 36 //g/ml of plasminogen. This fibrin gel was kept at 37°C for several hours to develop the spontaneous lysis with urokinase-activated plasminogen. Stability of the Inhibitor—To study the stability of the inhibitor towards a denaturing agent, affinity-purified inhibitor at various concentrations was incubated with 0.2% SDS for 30 min at 37'C. The SDS-treated inhibitor was renatured by mixed micelle formation with 2% Triton X-100 for 30 min at 23°C, and then the residual inhibitor activity was assayed using the two-step colorimetric assay. Control samples were treated with distilled water instead of SDS. The acid stability of the inhibitor was tested at pH 2.7 for 30 min at 37°C, by careful addition of cone. HC1 solution.

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culture in DMEM containing 10% fetal calf serum, 50 //g/ ml of ascorbic acid, 2 mM glutamate, 100 u/ml of penicillin, and 100 //g/ml of streptomycin at a cell density of 5 X 105 cells/cm2 in 17 mm wells (Lindbro multiwell plates, Flow Labs, McLean, Va.). On the third day the cell layer was washed three times with serum-free DMEM containing 0.3% gelatin to remove all traces of serum. Then the culture was maintained with serum-free DMEM containing 0.3% bovine serum albumin (essentially fatty acid-free, Sigma Chemical Co.), 50 // g/ml of ascorbic acid, 2 mM glutamate, 5 //g/ml of human transferrin, 100 u/ml of penicillin, and 100 //g/ml of streptomycin. The serum-free culture medium was subsequently changed every day and samples were kept at — 70°C after adding NaN3 to a final cone, of 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 showed round and polygonal morphology during the early stage of culture. To characterize cultured chondrocytes, the monolayer culture was labeled with 35S-inorganic sulfate (Amersham Australia, Sydney). After labeling, the culture medium and cell layer were separately analyzed for newly synthesized radio-sulfate labeled macromolecules (proteoglycans) after treatment with 4 M guanidine hydrochloride solution. The extracts were fractionated on a Sepharose 4B (Pharmacia Australia, North Ryde, N.S.W.) column under associative conditions. The radioactivity remaining after dialysis showed that 80% of radio-sulfated macromolecules were secreted into medium, and 20% were localized in the cell layer. Over 68% of radioactivity extracted from the cell layer was excluded at the void volume on Sepharose 4B gel chromatography (data not shown). These results suggested that the cultured chondrocytes had the ability to synthesize the aggregate form of proteoglycans and therefore retained some of their chondrocyte features at least in the early culture stage. Determination of Inhibitor Activity—PA inhibitor activity was assayed by measurement of the residual urokinase activity after preincubation with samples using the twostep 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, 0.2 Ploug units (PU)/ml; high-Mr urokinase: 80,000 PU/mg 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 5 mM 6-aminocaproic acid at 23°C for 90 min. Then 40 //I of affinity-purified human plasminogen (24) (50 //g/ml in the glycine buffer) was added and the mixture was incubated for 45 min at 37°C. The plasmin generated was assayed by the addition of thioesterase substrate (Z-lysine-thiobenzyl ester, Peninsula Laboratories, San Carlos, Calif.) and 1 ml of color agent (23). The mixture was further incubated for 30 min at 37°C, and the absorbance was read at 412 nm. One unit of urokinase inhibitor was defined as the amount of inhibitor sufficient to give 50% inhibition of 4 mPU of high-Mr urokinase in the two-step colorimetric assay. Plasmin (human, Sigma Chemical Co.) and trypsin (bovine pancreas, type III, Sigma Chemical Co.) inhibition was assayed using thioesterase substrate as in the second step of the urokinase inhibition assay. Thrombin (bovine, Sigma Chemical Co.) inhibition was assayed using Spectrozyme

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RESULTS

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

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Fig. 1. Titration of urokinase with chondrocyte culture medium. Day 3 chondrocyte culture medium was diluted with non-conditioned medium as indicated. Then 20u^ of diluted chondrocyte culture medium was incubated with high-Mr urokinase (4 mPU in 20 ^1) at 23*C for 90 min. The residual urokinase activity was assayed by the two-step colorimetric assay.

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

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1 2 3 4 Days after changing the medium to serum tree DMEM

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Fig. 2. Secretion of urokinase inhibitory activity by chondrocyte culture. Chondrocytes were inoculated in DMEM containing 10% fetal calf serum at a cell density of 5 x 105 cells/cm2. From the third day after inoculation, the cultures were harvested with serumfree DMEM containing 0.3% bovine serum albumin without cycloheximide (O) or with 3 pi g/ml of cycloheximide ( • ) . Serum- free culture medium (0.25 ml/cm2) was replaced every 24 h. Cell lysates of the culture without cycloheximide (A) and with cycloheximide (*) were extracted with 0.5% Triton X-100, 50 mM glycine pH 7.8 (0.25 ml/ cm2) at 4'C for 1 h.

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