Metalloproteinase and Activation of - Europe PMC

American Journal of Pathology, Vol. 151, No. 1, July 1997 Copynight X3 American Society for Investigative Pathology

Expression of Membrane-Type 1 Matrix Metalloproteinase and Activation of Progelatinase A in Human Osteoarthritic Cartilage

Kazushi Imai,* Satoru Ohta,*t Tadami Matsumoto,t Noboru Fujimoto,t Hiroshi Sato,§ Motoharu Seiki,§ and Yasunori Okada* From the Departments of Molecular Immunology and Pathologe and Molecular Virology and Oncology,5 Cancer Research Institute, and the Department of Orthopedic Surgery,t School of Medicine, Kanazawa University, Kanazawa, Ishikawa, and Fuji Chemical Industries Ltd.,t Takaoka, Toyama, Japan

Matrix metaloproteinases (MMPs) are expressed in osteoarthritic (OA) cartilage and are thought to be involved in the degradation of cartilage extracelular matrix (ECM). Among these proteinases, MMP-2 (gelatinase A) demonstrates a wide range of substrate specificity against the ECM present in cartilage. Although MMP-2 expression increases in OA cartilage, the activation mechanism of the corresponding zymogen (proMMP-2) in cartilage is unknown. In this study, we examined the expression pattern of membrane-type I MMP (MTI-MMP) in human OA articular cartilage and its correlation with the activation of pro-MMP-2. Immunohistochemical studies demonstrate that MTI-MMP localizes to the chondrocytes in the superficial and transitional zones in aU of the samples examined directly correlating with cartilage degradation. Reverse transcription polymerase chain reaction confirmed the predominant expression of MT1MMP mRNA in the OA cartilage. In situ hybridization revealed the site of expression of MT1MMP in OA cartilage to be the chondrocytes. Through gelatin zymography and a sandwich enzyme immunoassay it was demonstrated that OA cartilage explants secrete signiflcantly higher levels ofpro-MMP-2 than normal samples. Pro-MMP-2 activation was enhanced in the OA

cartilage samples and correlated with MT1-MMP expression in the cartilage. Plasma membranes preparedfrom cultured chondrocytes with MT1MMP expression and those directly isolatedfrom OA cartilage could activate pro-MMP-2. MT1MMP gene expression in cultured chondrocytes was induced by treatment with interleukin-1 a and/or tumor necrosis factor-ae. These data suggest that cytokine-induced MTI-MMP in the chondrocytes may play a key role in the activation of pro-MMP-2 in the OA articular cartilage, leading to cartilage destruction through ECM degradation. (Am JPathol 1997, 151:245-256)

Osteoarthritis (OA) is a common disease and increases progressively with age.1 It is characterized by loss of proteoglycans and collagens from the cartilage matrix, leading to fibrillation and eventually complete loss of cartilage exposing the underlying subchondral bone.2 Increased proteolytic activity in the joint is one of the possible mechanisms leading to cartilage destruction.3 As migration of inflammatory cells is minimal in the initial stage of the disease, proteinases derived from chondrocytes are most likely to be responsible for cartilage matrix degradation. Among the proteinases identified in chondrocytes, matrix metalloproteinases (MMPs) are thought to play a key role in the extracellular matrix (ECM) degradation of the cartilage.3 Supported by grant-in-aid 07457049 (to Y. Okada) and 5780 (to K. Imai) from the Ministry of Education, Science, and Culture of Japan. K. Imai was a recipient of Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists. Accepted for publication March 26, 1997. Address reprint requests to Dr. Yasunori Okada, Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920,

Japan.

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MMPs are zinc-dependent neutral endopeptidases with a wide spectrum of substrate specificity and consist of at least 14 different members. They can be subdivided into five groups according to their structural and enzymic properties: interstitial collagenases, gelatinases/type IV collagenases, stromelysins, membrane-type MMPs (MT-MMPs) and others.4 Among the MMPs, MMP-2 (gelatinase A) is important to complete collagen degradation after the specific cleavage of the triple helical region of the fibrillar collagen molecules by collagenases. MMP-2 also digests other substrates including aggrecan, link protein, decorin, fibronectin, and type X and XI collagens,5-10 all of which are components of the articular cartilage matrix. It is therefore likely that MMP-2 contributes to the cartilage matrix breakdown when produced in the articular cartilage. Actually, enhanced expression of pro-MMP-2 is reported in human OA articular cartilage.11 However, as most MMPs are secreted from cells as inactive zymogens (pro-MMPs), activation of pro-MMPs is another key step for the MMPs to function in vivo. Serine proteinases may be important in the activation of many pro-MMPs such as pro-MMP-1 (tissue collagenase), pro-MMP-3 (stromelysin 1), and pro-MMP-9 (gelatinase B).3 However, pro-MMP-2 is unique in that its activation is not caused by serine proteinases but achieved by membrane-associated activators.8'12 We have previously cloned MT1-MMP and identified it as an activator of pro-MMP-2.13 However, very little is known about MT1-MMP expression and its relation to the pro-MMP-2 activation in the cartilage. In the present study, we demonstrate for the first time that OA articular cartilage highly expresses MT1-MMP correlating with pro-MMP-2 activation whereas normal cartilage exhibits negligible MT1MMP expression and pro-MMP-2 activation. Plasma membrane fractions isolated from chondrocytes with MT1-MMP expression can activate pro-MMP-2. Both MT1-MMP expression and pro-MMP-2 activation in cultured chondrocytes are stimulated by treatment with interleukin (IL)-la and less efficiently by tumor necrosis factor (TNF)-a. Our data suggest that MT1 MMP inducible by the cytokines may contribute to the activation of pro-MMP-2 facilitating the ECM degradation in human OA. -

Materials and Methods Source and Histology of Articular Cartilages Non-osteophytic cartilage samples were obtained from 39 hip and 25 knee joints of 64 OA patients (mean age, 64 + 12 years) undergoing operations

for total hip or knee joint replacement. Normal control samples without macroscopic and microscopic changes were taken from 11 hip and 4 knee joints of 15 patients (74 + 9 years) with femoral neck fracture or an amputation operation due to atherosclerotic gangrene or malignant mesenchymal tumors. All of the samples were cut into slices (-3 mm thick), fixed with periodate-lysine-paraformaldehyde (PLP) or 4% paraformaldehyde fixatives for -24 hours at 40C, and embedded in paraffin wax after decalcification with 0.5 mol/L EDTA, pH 7.4. PLP-fixed paraffin sections (4 l,m thick) were stained with hematoxylin and eosin or toluidine blue and subjected to histological/ histochemical grading according to Mankin et al.14 The Mankin grading system, which indicates the morphological damage and loss of proteoglycans histochemically in articular cartilage, is used as an index of cartilage degradation.

Immunohistochemistry and in Situ Hybridization Sections from the PLP-fixed samples (64 from OA and 15 from normal cartilage) were treated with 0.3% H202 and 10% normal horse serum to block endogenous peroxidase and nonspecific binding, respectively. The sections were then treated with a monoclonal antibody to MT1-MMP (20 jig/ml, clone 1146G6; H. Ueno, H. Nakamura, K. Imai, H. Sato, M. Seiki, and Y. Okada, Cancer Res., in press), the antibody absorbed with purified recombinant MT1MMP15 or nonimmune mouse IgG (20 ,tg/ml). After reactions with biotinylated horse IgG to mouse IgG (Vector Laboratories, Burlingame, CA) and an avidin-biotin-peroxidase complex (DAKO, Glostrup, Denmark), the color was developed with 3,3'-diaminobenzidine tetrahydrochloride as described previously. 16 Immunostaining for MMP-2 was performed in a similar fashion using a monoclonal antibody to MMP-2 (2 ,ug/ml, clone 75-7F7).17 For in situ hybridization, the cDNA (177 bp) specific to MT1-MMP was prepared by polymerase chain reaction (PCR) using an antisense primer (5'TCGGCCCAAAGCAGCAGCTTC-3') and a sense primer (5'-CTTCATGGTGTCTGCATCAGC-3') and subcloned into Bluescript 11 KS ± (Stratagene, La Jolla, CA). Paraffin sections of the tissues fixed with 4% paraformaldehyde (six OA and three normal samples) were treated with 20 ,ug/ml proteinase K (Promega Biotec, Oakland, CA) and then 0.0025% acetic anhydride (Eastman Kodak, Rochester, NY) in 0.1 mol/L triethanolamine, pH 8.0 (Eastman Kodak), as described previously. 18 After prehybridization,

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the sections were hybridized with 35S-labeled antisense RNA or sense RNA for -12 hours at 500C. The slides were then treated with 20 ,tg/ml RNAse A, washed under stringent conditions (2X SSC, 0.5X SSC, and 0.1X SSC, twice each for 30 minutes at 500C), and air dried. They were dipped in NR-M2 emulsion (Konica, Tokyo, Japan) and subjected to autoradiography by exposing for 7 to 10 days.

Cultures of Cartilage Slices and Detection of MMP-2 Full-thickness cartilage slices with (18 cases) or without (7 cases) OA changes were cultured in serumfree minimal essential medium (MEM) containing 0.2% lactalbumin hydrolysate for 3 days. The culture media (6 ,ul/mg tissue dry weight) were subjected to gelatin zymography using SDS-polyacrylamide gels containing 0.2% gelatin. The intensity of the gelatinolytic bands corresponding to pro-MMP-2 of Mr 68,000 and active MMP-2 of Mr 62,000 was measured by computer-assisted image analysis according to the method of Davies et al,19 and the activation ratio (the ratio of the active MMP-2 form to proMMP-2 and active forms) was estimated. Pro-MMP-2 levels in the media were measured by a sandwich enzyme immunoassay specific to pro-MMP-2.17 Gelatinolytic activities were assayed in a solution assay using 14C-gelatin in the presence and absence of 1 mmol/L p-aminophenylmercuric acetate (APMA).8 The assay was performed in the presence of 2 mmol/L phenylmethylsulfonyl fluoride and 5 mmol/L N-ethylmaleimide to inhibit serine and cysteine proteinases. One unit of the activity degraded 1 ,tg of

gelatin/minute at 370C.

Isolation of Chondrocytes and Cell Culture Chondrocytes were obtained from OA or normal cartilages by digestion of the minced cartilages with 0.4% (w/v) Pronase (Calbiochem, La Jolla, CA) for 1 hour at 370C followed by 0.025% (w/v) bacterial collagenase type (Worthington Biochemical Corp., Freehold, NJ) for 12 to 16 hours at 370C.20 For monolayer cell cultures, isolated cells were plated on culture flasks at 5 x 104 cells/cm2 in MEM supplemented with 10% fetal bovine serum until the cells substantially reached confluence. The culture media of chondrocytes isolated from two OA cases were replaced with serum-free MEM containing 0.2% lactalbumin hydrolysate and then treated with 100 U/mI IL-ia (Dainippon Pharmaceutical Co., Osaka, Japan) and/or 500 U/ml TNF-a (Dainippon) for 36 hours.

Reverse Transcription (RT)-PCR Total RNA was isolated from chondrocytes (seven OA and four normal cartilage samples) using the acid guanidium-phenol-chloroform method.21 RNA extracted was treated with RNAse-free DNAse (Boehringer Mannheim, Mannheim, Germany) to eliminate DNA contamination in the samples and converted to a single-stranded cDNA using a random oligonucleotide hexamer (Takara, Otsu, Japan). Randomly primed cDNAs were prepared from 5 ,ug of total RNA by Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD) followed by PCR amplification. The primers prepared for in situ hybridization were utilized for the PCR amplification of MT1-MMP. As a control, for the PCR reaction, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified with a 5'-CCACCCATGGCAAATTCCATGGCA-3' (forward primer) and a

5'-TCTAGACGGCAGGTCAGGTCCACC-3' (reverse primer). PCR amplification was performed by running 30 cycles under the following conditions: denatured at 94°C for 30 seconds, annealed for 30 seconds at 560C for MT1-MMP or at 500C for GAPDH, and extended at 720C for 1 minute.

Northern Blotting Total RNA was extracted by the acid guanidiumphenol-chloroform method from primary cultures of OA chondrocytes (two cases) stimulated with 100 U/ml IL-la and/or 500 U/ml TNF-a in serum-free MEM for 36 hours. RNA (15 ,ug) was electrophoresed on 1.0% formaldehyde-agarose gels and transferred to nylon filters (Hybond-N, Amersham, Tokyo, Japan) followed by cross-linking with ultraviolet light. Filters were hybridized with 32P-labeled probes for the MT1-MMP gene as described previously.22

Preparation of Chondrocyte Membrane Fractions Cultured chondrocytes (three OA cases and one normal case) and chondrocytes directly isolated from four OA and two normal articular cartilages by the sequential enzymic treatment were homogenized in 10 mmol/L Tris/HCI, pH 7.5, containing 0.25 mol/L sucrose, 2 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L N-ethylmaleimide. The whole-cell homogenates were centrifuged at 3000 x g for 10 minutes to remove nuclei, and the cell organelleenriched fractions in the supernatants were pelleted by centrifugation at 100,000 x g for 90 minutes. The pellets were resuspended in 50 mmol/L Tris/HCI, pH

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7.5, 0.15 mol/L NaCI, 10 mmol/L CaCI2, 0.02% NaN3, layered onto a discontinuous sucrose density gradient (20, 30, 50, and 60% sucrose), and centrifuged at 100,000 x g for 3 hours. The plasma-membraneenriched fractions at the 30 to 50% sucrose interface were collected and pelleted again. The pellets were dissolved in the same buffer and protein concentrations were determined by reading absorbance at 595 nm using dye reagent (BioRad, Hercules, CA). All of the procedures after homogenization were carried out at 40C. As a control, plasma membranes were prepared in the same way from MDA-MB-231 cells from a human breast carcinoma cell line that is known to express MT1-MMP.23

Immunoblotting The plasma-membrane-enriched fractions prepared from the cultured chondrocytes (three OA cases and one normal case) were subjected to SDS-polyacrylamide gel electrophoresis (8.5% total acrylamide) under reducing conditions. Proteins separated in the gels were electrophoretically transferred onto nitrocellulose filters. The filters were then incubated for -12 hours at 230C with a monoclonal antibody specific to MT1-MMP (8 ,ug/ml; clone 114-1F2)13 and reacted with biotinylated horse IgG to mouse IgG, and the color was developed with 3,3'-diaminobenzidine tetrahydrochloride as described previously.16

Pro-MMP-2 Activation by Chondrocyte Plasma Membranes Pro-MMP-2 (2.6 ng) purified from human rheumatoid synovial cells8 was incubated with the plasma membranes (200 ng) from cultured chondrocyte (three OA cases and one normal case), those isolated directly from the articular cartilages (four OA and two normal cases), or MDA-MB-231 cell membranes in 20 ,ul of 50 mmol/L Tris/HCI, pH 7.5, 0.15 mol/L NaCI, 10 mmol/L CaCI2, and 0.02% NaN3 for 8 to 10 hours at 370C. The samples were subjected to gelatin zymography (8.5% total acrylamide), and the gels stained with Coomassie brilliant blue R-250.

Statistical Analyses Mann-Whitney U test was used to compare the data of OA and normal samples. Gelatinolytic activities in culture media with or without activation by APMA were compared using Wilcoxon single-rank test. Simple linear regression or Spearman rank correla-

tion was used for analyses of relationships between different parameters recorded in this study.

Results Immunolocalization and mRNA Expression of MT1-MMP in Osteoarthritic Cartilage OA cartilage samples varied in their histological appearance, ranging from surface irregularities to marked fibrillation and fissuring. The distribution of the histological/histochemical scores ranged from 2 to 13 with a mean score (±SD) of 8.2 ± 3.1. Normal articular cartilage had little or no microscopic changes with grades 0 to 1. By immunohistochemistry, the localization of MT1MMP was observed in all of the OA samples examined (64/64 cases). MT1 -MMP mainly localized to the chondrocytes in the superficial and transitional zones, where proteoglycans were depleted from the matrix (Figure 1 B). The chondrocytes located in the radial zone also stained for MT1-MMP when the cartilage had deep fissures reaching this zone (Figure 1C). Clustered chondrocytes close to the fissures were also labeled with the antibody (Figure 1 C). The immunostaining was considered to be specific for MT1-MMP as the staining was abolished with the monoclonal antibody absorbed with recombinant MT1 -MMP (Figure 1 D) and no staining was observed with nonimmune mouse IgG (data not shown). Approximately 50% of the total chondrocytes on average (48.1 ± 28.6%) immunostained positively in OA cartilage samples. In contrast, MT1-MMP staining was found in 27% of the normal cartilage samples (4/15 cases) but only in a few chondrocytes (5.2 ± 3.4%) in the superficial zone (Figure 1A). The percentage of MT1-MMP-positive chondrocytes was significantly higher in the OA samples than in the normal cartilage (P < 0.01). A linear correlation was found between the percentage of immunostained chondrocytes and Mankin score (p = 0.847; Figure 2). Immunolocalization of MMP-2 co-localized with the MT1-MMP-positive chondrocytes in almost all of the OA samples examined (K. lmai and Y. Okada, manuscript in preparation). In normal cartilage, chondrocytes in the superficial zone also reacted with the anti-MMP-2 monoclonal antibody. Expression of MT1-MMP mRNA in cartilage was examined by RT-PCR and in situ hybridization. RTPCR using a specific primer pair amplified a single 177-bp product in all of the OA cartilage samples examined (seven of seven cases), but the products were detected in only one of the four normal control


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Figure 1. Immunolocalization ofMT1-MMP in the normal and OA cartilages. Immunostaining was performed as described in Materials and Methods. A: Normal cartilage, Mankin grade 0. No staining is seen. B: OA cartilage, Mankin grade 4. Chondrocytes in the superficial and transitional zones stain (arrows). C: OA cartilage, Mankin grade 8. Chondrocytes in the transitional and radial zones strongly stain. Arrows indicate immunoreactive chondrocytes in clusters. D: OA cartilage (Mankin grade 5) stained with the monoclonal antibody absorbed with recombinant MT1-MMP. Bar, 100

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GAPDHD Figure 3. Determination of MT1-MMP mRNA expression in OA and normal cartilage by RT-PCR. Total RNA was extracted from OA (lanes 1 to 7) and normal (lanes 8 to 11 ) cartilage and reverse-transcribed into cDNA followed by a PCR reaction using specific primer pairs for MT1 MMP or GAPDH.

cartilage samples (Figure 3). The products were considered not to be derived from DNA contamination, as the template RNA was obtained from total RNA treated with RNAse-free DNAse and amplification did not occur when total RNA without the RT reaction was used for the PCR templates (data not shown). Primers specific to GAPDH generated a specific single amplicon of 598 bp without additional bands (Figure 3). In situ hybridization was carried out to identify the cells expressing MT1-MMP mRNA in the articular cartilage. Chondrocytes in the superficial and transitional zone of the OA cartilage were labeled with the antisense RNA (Figure 4A), whereas specimens from normal cartilage were not labeled (data not shown). The sense probe gave only a background signal in the chondrocytes of OA and normal cartilage (Figure 4B).

Production and Activation of Pro-MMP-2 in the Cartilage Culture media of the explants from 18 OA and 7 normal cartilage samples was subjected to gelatin zymography. Although the secretion of pro-MMP-2 of Mr 68,000 was observed in most of the samples examined, the intensity of the lytic band in the gels

Figure 4. In situ hybridization qfMTI-MMP in OA cartilage. Paraffin sections u'ere reacted uith -IS-labe(levd antisense (A) or .sense (B) riboprobes as described in Materials and Metbods. Note intense labeling in the chondrocytes in the s.uperficial zone of the articular cartilag,e uwith the antisenseprobe (arrows) and not with the sense probe. Bar, 25 ,um.


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Figure 6. Quantity ofpro-MMP-2 (A) and gelatinolytic activity (B) in the culture media secreted from cartilage explants. A: Culture media were obtainedfrom explants of normal (NOR; n = 7) and OA (n = 18) cartilages. Pro-MMP-2 was measured by a sandwich enzyme immunoassayforMMP-2. B: Gelatinolytic activity in the media from normal (n = 7) and OA (n = 18) cartilage was determined using "4C-gelatin solution assays in the absence (-) or presence (+) of APMA. Bars indicate mean + SD. 4P < 0.01; *¶ < 0.05 (Mann-Whitney U test).

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NIT1 inmmul o reactivities (%) Figure 5. Gelatin zymography of the culture media from cartilage explants (A) and correlation between pro-MMP-2 activation and MT1MMP immunoreactivity (B). A: Culture media were subjected to gelatin zymography as described in Materials and Methods. Seven representative OA samples (lanes 1 to 7) andfour normal cartilage samples (lanes 8 to 11 ) are shown. The major gelatinolytic activity of 68 and 62 kd corresponds to pro-MMP-2 and active MMP-2, respectively. ProMMP-9 with 92 kd is also observed in two OA samples (lanes 2 and 6). Protein standards include phosphorylase b (94 kd), human transferrin (77 kd), BSA (68 kd), IgG heavy chain (55 kd), ovalbumin (43 kd), and carbonic anhydrase (29 kd). B: A linear increase in the activation ratio of pro-MMP-2 with MT1-MMP immunoreactivity is found by simple linear regression (r2 = 0.680; n = 18).

appeared to be higher in the OA samples than in the normal cartilage samples. Importantly, the active MMP-2 species of Mr 62,000 was detected in all of the samples from OA cartilage, whereas normal samples produced a negligible amount of active MMP-2 (Figure 5A). In fact, the activation ratio of pro-MMP-2 (the ratio of the active MMP-2 to proMMP-2 and active forms) was significantly higher in the OA cartilage samples (41 ± 16%) than in the normal samples (13 ± 8%; P < 0.01). The proportion of the activation ratio demonstrated a linear dependence on the MT1-MMP immunoreactivities of chondrocytes (r2 = 0.680; Figure 5B). There was also a linear correlation between the activation ratio and the

Mankin scores (p = 0.664; data not shown). ProMMP-9 of Mr 92,000 and the processed form of Mr 83,000 were detectable in 28% of the OA samples (5/18 samples) but not in the normal (0/7 samples). As gelatin zymography demonstrated enhanced production and activation of pro-MMP-2 in the OA cartilage, we further quantified the amounts of proMMP-2 protein secreted in the culture media by a sandwich enzyme immunoassay for pro-MMP-2 and gelatinolytic activities in a solution assay using 14C_ gelatin. As shown in Figure 6A, the amount of proMMP-2 in the media from OA cartilage (10 + 7 ng/mg tissue dry weight) was significantly higher than that from normal cartilage (2 ± 1 ng/mg tissue dry weight). Gelatinolytic activities assayed in the absence of APMA were also remarkably higher in the OA samples (38 + 30 U/g tissue dry weight) than in the normal samples (3 ± 4 U/g tissue dry weight; Figure 6B). The presence of APMA in the assay significantly increased the activity in the normal cartilage samples (7 + 3 U/g dry weight; P < 0.05) without a definite change of activity observed in the OA samples, although the level (42 ± 26 U/g dry weight) was remarkably higher than that of the APMA-treated normal samples (Figure 6B). These data are consistent with the finding of the gelatin zymography demonstrating that the OA cartilage has an enhanced production of MMP-2 with a large proportion of the enzyme activated, in contrast to the limited production and activation in normal cartilage. An increase in the pro-MMP-2 activation ratio did not occur with pro-MMP-2 secretion (data not shown), suggesting that the activation and the production of pro-MMP-2 are two independent events.

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~~~29 Figure 7. Expression ofMT1-MMP and pro-MMP-2 activation by chondrocyte membranes. A: Cell membranes (3.2 gg) prepared from MDAMB-231 cells (lane 1), cultured normal chondrocytes (lane 2), and OA chondrocytes (lanes 3 to 5) were subjected to immunoblotting for MT1-MMP as described in Materials and Methods. B: Pro-MMP-2 was incubated for 8 hours at 3 70C with buffer alone (lane 1) or cell membranes prepared from MDA-MB-231 cells (lane 2), cultured nor-

mal chondrocytes (lane 3), and OA chondrocytes (lanes 4 to 6), and the generation of active MMP-2 utas demonstrated by gelatin zymography (8.5% total acrylamide). C: Pro-MMP-2 activation with cell membranes directly isolatedcfrom normal (lane 2) and OA (lanes 3 and 4) cartilage u'as examined by a 10-houir incubation at 37°C as described above. Lane 1 is pro-MMP-2 incuibated utith buffer alone. Note that cell membranes from both normal and OA chondrocytes in cuilture contain MT1-MMP and activate pro-MMP-2, whereas activation is seen only in chondrocyte membranes isolated directly from OA cartilages.

Activation of Pro-MMP-2 by Chondrocyte Membranes Immunoblotting analyses demonstrated that, under the culture conditions, chondrocytes from one normal and three OA cartilage samples expressed MT1MMP protein of Mr 60,000, which corresponds to activated MT1-MMP on the cell membranes23 (Figure 7A). RT-PCR also demonstrated that MT1-MMP mRNA is unambiguously expressed in those chondrocytes (data not shown). When cell membrane fractions were prepared from these cultured chondrocytes and incubated with pro-MMP-2 of Mr 68,000, the zymogen was processed into the active species of Mr 62,000 (Figure 7B). Similar pro-MMP-2 activation was obtained with plasma-membrane-enriched fractions of the chondrocytes directly isolated from OA cartilage with MT1-MMP expression, but such activation was not observed with the chondrocyte membranes from the normal cartilage (Figure 7C).

Effects of IL- 1 a and/or TNF-a on MT1 -MMP Gene Expression and Pro-MMP-2 Activation in Chondrocytes As IL-la and TNF-a are commonly present in OA cartilage, their effects on MT1-MMP mRNA expression were examined by Northern blot analysis. Although chondrocytes without cytokine treatment ex-

Figure 8. M71-MMP mRNA expression (A) andpro-MMP-2 activation (B) in cultuired OA chondrocytes treated with IL-la and/or TNF-a. A: Chondrocytes in culture were treated with serum-free medium alone (lane 1), 100 Ulml IL-la (lane 2), 500 Ulml TNF-a (lane 3), or 100 Ulml IL-la and 500 Ulml TNF-a (lane 4) as described in Matenials and Methods. Total RNA ( 15 jig) from eacb sample separated on a formamidelformaldehyde agarose gel was blotted onto nylon membranes that were probed with MT1-MMP cDNA. The 18 S rRNA was stained with ethidium bromide for comparison of the total RNA loaded. B: Culture media (30 gIl/lane) were harvested from the chondrocytes treated as described above and subjected to gelatin zymography (8% total acrylamide). Small and large arrows indicate pro-MMP-2 and active MMP-2, respectively. The arrowhead shows pro-MMP-9.

pressed a low level of MTl-MMP mRNA, both IL-la and TNF-a tremendously enhanced the expression of MTl-MMP mRNA (Figure 8A). The stimulatory effect of IL-la was 2.5- to 8-fold higher than TNF-a. In addition, the stimulation with both IL-la and TNF-a was not synergistic. Culture media were harvested from the chondrocytes and subjected to gelatin zymography. There was no definite difference in the secreted pro-MMP-2 levels among the chondrocyte cultures with or without the cytokines. However, it is notable that activation of pro-MMP-2 was enhanced by IL-la and TNF-a, the latter of which also upregulated the pro-MMP-9 production as shown with the HT1080 human fibrosarcoma cell line.24

Discussion Our studies demonstrate for the first time that MT1MMP is highly expressed in the chondrocytes of OA articular cartilage. Immunolocalization studies demonstrated that approximately 50% of the chondrocytes located in the superficial and transitional zones stained for MTl-MMP in all of the OA samples examined with only a few chondrocytes in the superficial zone of normal cartilage positive for MTl-MMP. RT-PCR confirmed the predominant MTl-MMP expression in OA cartilage. Also, in situ hybridization with RNA probes demonstrated that


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OA chondrocytes are the cells expressing MT1MMP mRNA. Previous studies have reported that OA chondrocytes express various MMPs including MMP-1, MMP-2, MMP-3, MMP-8 (neutrophil collagenase), MMP-9, and MMP-13 (collagenase 3).32i27 The present results add MT1-MMP to the list of MMPs produced in the OA cartilage. The previous studies by Mohtai et al11 reported that expression of both MMP-2 and MMP-9 is enhanced in OA cartilage, although they emphasized the role of MMP-9 in the progressive cartilage degradation in OA. In the present studies, increased production of pro-MMP-2 from the OA cartilage explants was ascertained by gelatin zymography. A sandwich enzyme immunoassay specific to proMMP-2 also indicated that the production level of MMP-2 is approximately fourfold higher in OA cartilage samples compared with normal cartilage. Furthermore, we showed that activation of pro-MMP-2 in the cartilage is accelerated by the OA changes, demonstrating a linear correlation between the activation ratio and Mankin score. Recent molecular cloning and experimental studies of MT1-MMP have contributed to the understanding of the activation mechanism of pro-MMP-2. The activation is considered to be initiated by direct cleavage of the Asn37Leu38 bond of pro-MMP-2 by MT1-MMP and completed by intermolecular autocatalysis of MMP2.28 30 It is also established that, once MT1-MMP is synthesized within the cells, it is expressed in the active form of Mr 60,000 on the cell surface after being processed by furin.2331 Involvement of MTlMMP in the in vivo activation of pro-MMP-2 has been described in various human cancer tissues such as stomach and lung carcinoma.16'32 The present studies demonstrated a linear correlation between the pro-MMP-2 activation and MT1-MMP immunostaining in the OA cartilages. In addition, the chondrocyte membranes isolated directly from the OA cartilage activated pro-MMP-2, whereas no such activation was found in the normal cartilage membrane. Under the present culture conditions, the normal chondrocytes also expressed MT1-MMP. This is not surprising as chondrocytes cultivated on the flasks are modulated to change their phenotype,33 resulting in the MT1-MMP expression. Thus, these data suggest that MT1-MMP expression in the chondrocytes may be involved in the activation of pro-MMP-2 in OA cartilage. In the OA cartilage, degenerative changes start from the superficial layer and progress into the deeper zones.34'35 Previous immunolocalization studies on MMP-336 and MMP-837 demonstrated that the expression levels correlate with the severity of

OA changes in the cartilage. An identical correlation is seen in this study with MTl-MMP expression in the cartilage. As gene expression of many MMPs such as MMP-1 and MMP-3 is regulated by various cytokines and growth factors,4 enhanced MT1-MMP expression in the OA cartilage may be due to stimulation by such factors. MTl-MMP mRNA expression is known to be up-regulated by concanavalin A, phorbol myristate acetate, basic fibroblast growth factor, and TNF-a and down-regulated by glucocorticoids in various cells.28'38-41 However, the effects of these cytokines and growth factors on the expression of MMPs depend on the cell type used for the experiments, and information is not available on factors modulating MTl-MMP expression in human chondrocytes. Lohi et al40 have recently reported that MTl-MMP mRNA expression is enhanced by TNF-a in HT1080 fibrosarcoma cells but not in embryonic lung fibroblasts, and IL-1,B has no effect on the expression of MTl-MMP in these cells. In the present study, we demonstrate that both TNF-a and IL-la stimulate OA chondrocytes to express the MTl-MMP gene. Gelatin zymography further showed that MT1MMP up-regulated by the cytokines functions to activate pro-MMP-2. As both cytokines are major mediators of joint damage in OA cartilage,42 it seems likely that MTl-MMP expression in OA cartilage is controlled by these cytokines, which also up-regulate expression of MMP-1 and MMP-3. The regulation of MMP-2 gene expression is different from other MMPs in that MMP-2 is not inducible by many cytokines and growth factors such as IL-la and TNF-a 3 but is expressed constitutively in many cells in culture. In both normal and OA cartilage, MMP-2 was immunolocalized in many more chondrocytes than MTl-MMP (K. Imai and Y. Okada, manuscript in preparation). Our preliminary studies also show that in OA cartilage chondrocytes express MT3-MMP, but the expression is found only in the advanced disease cartilage and the mRNA is quite low (K. Imai and Y. Okada, unpublished data). It might be possible to speculate that MT3-MMP also accelerates the cartilage degradation through pro-MMP-2 activation in advanced OA cartilage but plays a minor role when compared with MT1-MMP. Expression of MT2MMP was not detectable in the OA cartilage examined (K. Imai and Y. Okada, unpublished data). Thus, the data suggest that MTl-MMP expression is a key determinant for MMP-2 activity in the cartilage through activation of the zymogen by MTl-MMP. Hollander et al34 showed by immunohistochemistry that type 11 collagen breakdown in OA cartilage is first observed around the chondrocytes. Similar degradation of type 11 collagen on the cell surface is also

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described when the normal articular cartilage is cultured in the presence of IL-1.4344 In addition to collagen degradation, aggrecan breakdown in cartilage is also thought to be a cell-membrane-dependent event.45 These data suggest that pericellular ECM breakdown is a key process that is occurring in OA cartilage. Recent experimental studies demonstrate that when MT1-MMP is expressed on the surface of cells pro-MMP-2 is captured and activated on the cell surface.29 As MT1-MMP and MMP-2 co-localized in the OA chondrocytes, MMP-2 activated by MT1-MMP may attack various ECM macromolecules such as aggrecan around the chondrocytes. In addition, recent biochemical studies on MMP-13 (collagenase 3) suggests that MT1-MMP also activates pro-MMP-13.46 As MMP-13 is expressed in OA cartilage and readily digests type 11 collagen,25'27 intermolecular activation of pro-MMP-13 by MT1-MMP may be important in the ECM breakdown in OA cartilage. On the other hand, it has been reported that MT1-MMP per se is an ECM-degrading enzyme that digests laminin, fibronectin, vitronectin, dermatan sulfate proteoglycan, and gelatin.31 We have also recently demonstrated that MT1-MMP is active against aggrecan and interstitial collagens, including type II collagen, cleaving them at the triple helical region similar to the cleavage by interstitial collagenases.15 Aggrecan fragments released from the OA cartilage matrix demonstrate the special NH2 terminus beginning at Ala374, which is thought to be generated by the action of so-called aggrecanase.47 It has been reported that the aggrecanase activity is inducible by IL-l a48 and inhibited by metalloproteinase inhibitors.49 Among the MMPs, only MMP-8 at high concentrations cleaves the Glu373-Ala374 bond of aggrecan, 5 but its expression level in the OA cartilage is low.25 Therefore, the possibility that MMP-8 is an aggrecanase is still speculative. On the other hand, as MT1-MMP is expressed on the cell surfaces and the gene expression is stimulated by treatment of the chondrocytes with IL-la, it is plausible to speculate that MT1-MMP may be responsible for the so-called aggrecanase activity.

Acknowledgments We are grateful to Dr. G. 1. Goldberg (Division of Dermatology, Washington University, School of Medicine, St. Louis, MO), a visiting professor at Kanazawa University, for helpful discussion and to Dr. J. D'Armiento (Department of Medicine, Columbia University, New York, NY) for critical reading of the manuscript. We also thank M. Takegami and S. Makino for their technical assistance.

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