We performed Southwestern screening as previously described, with a slight modification (53). The Escherichia coli XL1-Blue MRF′ strain was infected with the lambda phage library and then spread on NZY agar plates containing 0.5 M isopropyl-β-d-thiogalactopyranoside (IPTG). Formed plaques were lifted onto nitrocellulose membranes (GE Healthcare, Uppsala, Sweden). The double-stranded DNA (dsDNA) of four tandem repeats of TRENDIC was prepared by annealing the following oligonucleotides: 4× TRENDIC-s, 5′-ACG CGT [(CTG TGA GCT GGA GTG TGC CAGC) CAG]₄ CTC GAG-3′; 4× TRENDIC-as, 5′-CTC GAG [CTG (GCT GGC ACA CTC CAG CTC ACAG)]₄ ACG CGT-3′. The annealed probe was end labeled with [γ-³²P]ATP and was applied to the membranes in binding buffer (10 mM HEPES, pH 7.9, containing 50 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol [DTT], 0.25% skim milk, and 50 μg/ml of denatured and undenatured salmon sperm DNA). After the membranes had been washed three times with a wash buffer (10 mM HEPES, pH 7.9, containing 250 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, and 0.25% skim milk), they were exposed to X-ray films. The positive plaques were picked up for subsequent secondary and tertiary screenings as well. The final cDNA clones in a pBK-cytomegalovirus vector were obtained by in vivo excision using the E. coli XLOLR strain.

The cDNA cloned into each plasmid was sequenced by the dideoxy chain termination method (42) with a BigDye Terminator cycle sequencing ready reaction kit, version 2.0 (Applied Biosystems, Foster City, CA), and an ABI PRISM 310 genetic analyzer (Applied Biosystems). The sequences were analyzed by using BLASTn, Evidence Viewer, and UniGene online services at the NCBI website (http://www.ncbi.nlm.nih.gov/). These data on the cloned genes are summarized in Table 1.

The following human-derived cells were used: human chondrosarcoma-derived chondrocytic HCS-2/8 cells (40, 51), MDA-MB-231 breast carcinoma cells, HeLa cells derived from human cervical cancer, and SaOS-2 osteosarcoma-derived cells. A COS7 monkey kidney-derived cell line also was used. Cells were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in humidified air containing 5% CO₂ at 37°C. For immunocytochemistry and internalization studies, glass chamber slides (4 or 8 well) were coated with 50 μg/ml of collagen (Cellmatrix type I-C; Nitta-Gellatin, Osaka, Japan) for 30 min before cells were seeded.

We used the following anti-MMP3 antibodies: anti-MMP3 N-terminal region (5052; Sigma, St. Louis, MO), anti-MMP3 hinge region (4802; Sigma), anti-MMP3 C-terminal domain (4927; Sigma), and anti-MMP3 catalytic (CAT) domain antibody (4190; Sigma). We also used anti-CCN2/CTGF (AF660; R&D), anti-histone H3 (06-599; Upstate), anti-Sox6 (S7193; Sigma), anti-α-tubulin immunoglobulin G₁ (IgG₁) (T9026; Sigma), anti-cathepsin D (C-20; Santa Cruz Biotech, Santa Cruz, CA), anti-lamin A/C monoclonal IgM (sc-7293; Santa Cruz), and anti-β-actin (AC-74; Sigma). We used the following antibodies to detect tags: anti-Flag M2 (Sigma), anti-Myc tag (Abcam, Cambridge, United Kingdom), and anti-glutathione S-transferase (anti-GST) (GE Healthcare) antibodies. An antidigoxigenin alkaline phosphatase-conjugated Fab (Roche, Basel, Switzerland) was used in electrophoresis mobility shift assays (EMSA). For Western blotting, we used horseradish peroxidase-conjugated secondary antibodies against anti-mouse IgG (Amersham) and anti-rabbit IgG (Dako, Copenhagen, Denmark). An anti-rabbit IgG rhodamine conjugate (Sigma) was used for immunostaining. We used these antibodies at the concentrations instructed by the manufacturer. The MMP3 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Daiichi Pure Chemicals (Tokyo, Japan).

The nuclear and cytoplasmic proteins were prepared by using a CelLytic NuCLEAR extraction kit (Sigma) according to the manufacturer's protocol. The subcellular fractions were prepared by using a ProteoExtract subcellular proteome extraction kit (Calbiochem, San Diego, CA) according to the manufacturer's protocol. Total cell lysate was prepared by using a CelLytic M reagent (Sigma) according to the manufacturer's protocols. The protease inhibitor cocktail (Sigma) was added at the appropriate steps.

Extracted proteins were heated at 95°C for 5 min in sodium dodecyl sulfate (SDS) sample buffer in the presence of 5% 2-mercaptoethanol and separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 12% polyacrylamide gel. Alternatively, proteins were heated at 70°C for 10 min in lithium dodecyl sulfate sample buffer containing 50 mM DTT and were separated in a 10% Bis-Tris NuPAGE gel (Invitrogen) in morpholinepropanesulfonic acid running buffer containing an antioxidant. Semidry electroblotting was carried out using a polyvinylidene difluoride membrane (Hybond P; GE Healthcare). The membrane was blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% skim milk for 30 min at room temperature (RT). After being blocked, the membrane was incubated with the primary antibody overnight at 4°C and subsequently was incubated with the secondary antibody for 1 h at RT in the blocking solution. The blot was visualized by using an enhanced chemiluminescence (ECL) Western blotting analysis system (GE Healthcare) with chemiluminescence detection. The photogram was obtained by autoradiography or by using an ECL minicamera (GE Healthcare). The band signals obtained by Western blotting were quantified by using ImageJ, version 1.37 (Wayne Rasband, NIH).

A recombinant human proenzyme MMP3 (rhMMP3; R&D) was purchased and used for the molecular weight control and the internalization assay. For other experiments, we used our purified rhMMP3, prepared as described below. Organomercurial (4-aminophenyl)mercuric acetate (APMA) (Sigma) was dissolved in 50 mM NaOH. Active MMP3 was prepared by incubating proenzyme MMP3 (final concentration, 83.3 nM) with 1 mM of APMA in Tris buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl₂ and 0.05% Triton X-100) at 37°C for 4 h. All MMP inhibitors were purchased from Calbiochem and were dissolved and stored in dimethyl sulfoxide (DMSO). Before use, they were diluted 100-fold once in a cell culture medium (1% DMSO) and then added to cultured cells (final concentration, 0.01% DMSO). Transforming growth factor β (TGF-β) (10 ng/ml) was purchased from Peprotech (Rocky Hill, NJ).

The stromelysin endopeptidase activity assay was carried out by using the fluorescence resonance energy transfer peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ fluorogenic substrate II (R&D, Minneapolis, MN) (32). The substrate (10 μM) was mixed with enzyme or nuclear extract in a Tris buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl₂ and 0.05% Triton X-100) and incubated at 37°C. The relative fluorescence units (RFUs) of the reactant in a 96-well black plate were measured by using a Fluoroskan AscentFL (Labsystem, Helsinki, Finland).

For immunocytochemistry, cells were fixed in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, washed in TBS, and permeabilized with 0.2% Triton X-100 for 15 min. Incubation with primary and secondary antibodies was performed in PBS containing 1.5% normal goat serum and 0.05% Triton X-100 at RT for 1 h, respectively. After three washes with TBS, the mounting and DNA staining were performed by using ProLong gold antifade reagents with 4′,6-diamino-2-phenylindole (DAPI) (Molecular Probes, Invitrogen). Cells were observed with an immunofluorescence microscope (Biozero; Keyence, Osaka, Japan) or a Radiance 2100 laser scanning system (Bio-Rad).

For the conjugation of Cy3 to rhMMP3, the Tris-based solvent of rhMMP3 was replaced with sodium carbonate-sodium bicarbonate buffer (pH 9.3) by use of a Microcon YM-30 (Millipore, Billerica, MA), and the concentration was adjusted to 100 μg/ml. MMP3 (50 μl) was added to a vial of Cy3 monoreactive dye (GE Healthcare) and was incubated at RT for 30 min with mixing every 10 min. The labeled MMP3 was desalted and separated from the unconjugated Cy3 on a MicroSpin G-25 column (GE Healthcare) preequilibrated with PBS.

The cells in chamber slides were washed once with prewarmed serum-free medium and incubated with Cy3-MMP3 (1 μg/ml) in the serum-free medium at 37°C for 5, 10, 15, 30, or 60 min and subsequently were fixed with 4% paraformaldehyde in PBS for 15 min. The cells were stained with 10 nM Sytox green nucleic acid stain (Molecular Probes, Invitrogen) in TBS for 20 min and mounted in ProLong antifade reagents (Molecular Probes, Invitrogen). The cells were observed by confocal laser microscopy as described below, and Cy3-positive cells were classified into three groups: cells with cytoplasm-dominant signals, cells with both cytoplasmic and nuclear signals, and cells with nucleus-dominant signals.

Confocal laser scanning was carried out with a Radiance 2100 laser scanning system equipped with an argon and krypton laser (excitation wavelengths of 488 and 568 nm) (Bio-Rad) through an Eclipse TE2000-U microscope (Nikon) with a ×60 objective lens. The scan was visualized with the software Laser Sharp 2000 (Bio-Rad).

Nuclear localization signals (NLSs) in MMP3 were predicted by the PSORTII program (http://psort.nibb.ac.jp/helpwww2.html#src) (34). Several arginine- and lysine-rich sequences in MMP3 also were selected for analysis. pEGFP-NLSs were constructed as described below. COS7 cells were cultured in the 4-well chamber slides and were transfected with 200 ng of a series of pEGFP-NLS constructs by the aid of Fugene 6 transfection reagent (Roche). After being cultured for 24 h, cells were fixed with 4% formaldehyde in PBS and mounted with a fluorescent mounting medium (Dako). The subcellular localization of the enhanced green fluorescent protein (EGFP) was visualized with confocal laser scanning microscopy.

The chromatin immunoprecipitation (ChIP) assay was carried out according to the manufacturer's protocol (Upstate) with a slight modification. A half million HCS-2/8 cells were seeded in each well of a 6-well plate and were cultured for 48 h with a medium change at 24 h. Formaldehyde (final concentration, 1%) was added to the medium, and the cells were incubated for 10 min at 37°C. The cells were washed twice and scraped in ice-cold PBS containing a protease inhibitor cocktail (Sigma) and then centrifuged for 4 min at 200 × g at 4°C. Subsequently, the cells were lysed in 200 μl of SDS lysis buffer (50 mM Tris-HCl, pH 8.1, containing 10 mM EDTA and 1% SDS) for 10 min on ice. DNA was sheared by three 10-s rounds of sonication on ice by using a Handy Sonic model UR-20P (Tomy Seiko, Tokyo, Japan) at 30% of maximum power. After centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was diluted 10-fold in ChIP dilution buffer (16.7 mM Tris-HCl, pH 8, containing 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, and protease inhibitor cocktail), and 1% of it was retained as the input. The sample was precleared by rotating it with 80 μl (4%) of a 50% salmon sperm DNA-50% protein A-agarose slurry (Upstate) for 30 min at 4°C. After a brief centrifugation, the supernatant was rotated with or without the anti-MMP3 CAT domain antibody (1:500; Sigma) overnight at 4°C and was further incubated after adding 60 μl (3%) of the 50% salmon sperm DNA-50% protein A-agarose slurry at 4°C for 1 h. The protein A-agarose-antibody-antigen complex was centrifuged at 300 × g at 4°C for 1 min, and the pellet was washed once with low-salt wash buffer (20 mM Tris-HCl, pH 8.1, containing 150 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton X-100), once with high-salt wash buffer (20 mM Tris-HCl, pH 8.1, containing 500 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton X-100), once with LiCl wash buffer (10 mM Tris-HCl, pH 8.1, containing 0.25 M LiCl, 1% NP-40, 1% deoxycholate, and 1 mM EDTA), and twice with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The antigen complex was eluted by being mixed and rotated in 250 μl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO₃). The elution step was repeated, and the eluates were combined. A 4% volume of 5 M NaCl was added to the eluate and to the input sample. The cross-linked chromatin complex was reversed by being heated at 65°C for 4 h, and DNA was purified using QIAquick spin columns (Qiagen, Hilden, Germany). PCR was carried out using Prime STAR HS DNA polymerase (Takara) according to the manufacturer's protocol. Ten percent of the total purified DNA was used for the PCR in 50 μl of reaction mixture. The 204 bp of CCN2 enhancer fragment between −292 and −88 was amplified by using the primer pair Seq2-s (5′-GAA TCA GGA GTG GTG CGA AG-3′) and 110bp-as (5′-ATT CCT CGC ATT CCT CCC CAC CT-3′) in 30 cycles of PCR under the following conditions: 98°C for 10 s, 62°C for 30 s, and 72°C for 30 s. The 215-bp cDNA of GAPDH was amplified by using the primer pair GAPDH NEO LCL (5′-GCC AAA AGG GTC ATC ATC TC-3′) and GAPDH NEO LCR (5′-GTC TTC TGG GTG GCA GTG AT-3′) in 30 cycles of PCR under the following conditions: 98°C for 10 s, 65°C for 20 s, and 72°C for 20 s. The PCR products were analyzed by 2% agarose gel electrophoresis.

Cationic liposome-mediated DNA transfection was carried out with a Fugene 6 transfection reagent according to the manufacturer's optimized methodology (Roche). For reporter gene assays, cells were seeded in 12-well plates and cultured for 12 to 24 h. Subsequently, cells were transfected with a total of 1 μg of plasmid DNA in the reporter/effecter/control ratio of 2:1:1 or 10:10:1. Medium was changed 20 h after the transfection. After being cultured for a further 24 h, cells were lysed in 200 μl of 1× passive lysis buffer (Promega, Madison, WI) with gentle rocking for 20 min and were collected. Luciferase assays were carried out by using a Dual Glo luciferase assay system (Promega), as described previously, on a smaller scale (17). Relative luciferase activities were calculated as the ratios of firefly luciferase activity to Renilla luciferase activity.

Reverse transcription was carried out with 500 ng of the cytoplasmic RNA by using Omniscript reverse transcriptase (Qiagen) and oligo(dT) according to the manufacturer's protocol. The real-time PCR was carried out as described previously (7) using a LightCycler system (Roche) with Sybr green (Toyobo) according to the manufacturer's directions. The PCR conditions were as follows: primary denaturation at 95°C for 30 s, followed by 45 cycles of PCR at 95°C for 5 s, 65°C (for MMP3 and CCN2) or at 60°C (for GAPDH) for 10 s, and 72°C for 15 s. The signals were collected at 72°C in every cycle. The specific primers for real-time PCR were designed and synthesized by NGRL (Sendai, Japan) as MMP3LCL (5′-CAG GCT TTC CCA AGC AAA TA-3′) and MMP3LCR (5′-GTG CCC ATA TTG TGC CTT CT-3′) for human MMP3. The nucleotide sequences of the primer pairs for CCN2 and GAPDH were previously described (7).

The anti-MMP3 antibody affinity columns were prepared by using antibodies described above and a ProFound mammalian coimmunoprecipitation kit (Pierce, Rockford, IL) according to the manufacturer's protocol. Eluate (20% of the total) was used for the subsequent tryptic digestion using an in-solution tryptic digestion and guanidination kit (Pierce). The eluted proteins were reduced in ultrapure water containing 5 mM DTT and 25 mM ammonium bicarbonate at 95°C for 5 min. To minimize disulfate bond formation and side chain modification, the alkylation of the samples was carried out by adding iodoacetamide (IAA; 10 mM at final concentration) at RT for 20 min in the dark. The protein samples were digested by adding activated trypsin (final concentration, 3 ng/μl) and incubating them at 30°C overnight. Formic acid was added (final concentration, 0.1%). One microliter of the peptide samples was applied to nanospray high-performance liquid chromatography chip-ion trap-mass spectrometry/mass spectrometry (MS/MS) with an Agilent 1100 liquid chromatography (LC)/MSD Trap XCT Ultra series system (Agilent Technologies, Santa Clara, CA). The MS and MS/MS data were analyzed by the data analysis software Spectrum Mill, version 3.3 (Agilent). The cutoff score for proteins was 8.0. The score was defined based on the covering rate for amino acid sequences and the frequency of detected fragments. To remove the background signals, we excluded the data obtained with a control IgG column from those obtained with the anti-MMP3 columns.

Recombinant proMMP3, GST, GST-fused HP1α, HP1γ, NF45, NCoR1(1-152), and CAFp48/RBBP4 were prepared by a cold shock system (Takara) according to the manufacturer's protocol. These recombinant proteins were designed to be fused with a trigger factor (Takara), which improves protein solubility. E. coli Rosetta2(DE3)pLysS-competent cells (Invitrogen) were transformed by the pCold-derived vectors described above. Transformed clones were cultured in 2 ml of Luria-Bertani broth containing 500 μg/ml of carbenicillin at 37°C for 8 h. Cells were further cultured in 10 to 500 ml of the broth at 37°C until an optical density at 600 nm of between 0.4 and 0.5 was reached. The induction of the protein synthesis was carried out by cooling the E. coli at 15°C for 30 min and further shaking the sample for 24 h at 15°C after the addition of 0.5 mM IPTG. Cellular pellets were obtained by centrifugation, frozen at −80°C, melted, suspended in appropriate volumes of lysis buffer (50 mM Tris, pH 8.0, containing 500 mM NaCl, 1% Triton X-100, 1 μM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride [PMSF]), disrupted by four 30-s sets of sonication, and centrifuged at 17,000 × g for 15 min at 4°C. To confirm the recombinant protein production in the supernatants, we carried out SDS-10% PAGE and Coomassie brilliant blue (CBB) staining using CBB R-250 (Sigma). Bovine serum albumin (BSA) was used as a concentration standard.

The GST pull-down assay was carried out by using the soluble fractions including recombinant proMMP3, GST, and GST-fused proteins as described above. Twenty microliters of glutathione-Sepharose 4B beads (GE Healthcare) was washed once with 500 μl of the Tris buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) with protease inhibitors (0.5 mM PMSF and 1 μM pepstatin A). The beads were mixed with the soluble fractions containing 3, 10, or 30 μg of GST-fused recombinant proteins in 500 μl of the Tris buffer, and the soluble fraction containing 10 μg of recombinant proMMP3 tagged with Flag₃ was added. The mixture was rotated for 2 h at 4°C, and the precipitant was washed four times with 500 μl of the Tris buffer. The precipitant was dissolved in 20 μl of 2× SDS sample buffer containing β-mercaptoethanol, was boiled at 95°C for 5 min, and was separated by SDS-10% PAGE. Western blot analysis was performed using an anti-Flag M2 antibody (Sigma) or anti-GST antibody (GE Healthcare).

The hind limbs of 8-week-old female BALB/c nu/nu mice (Clea, Tokyo, Japan) were fixed with 10% neutral phosphate-buffered formalin. The experimental osteoarthritis was induced by injecting monoiodoacetic acid (MIA; Sigma) into the intraarticular spaces of 6-month-old Wister rats, as described previously (36). These procedures were approved by the Animal Committee of the Okayama University Dental School. The animals were processed for histological analysis at 6 weeks after the injections. The specimens were decalcified in a 14% EDTA solution for 2 to 3 weeks and then embedded in paraffin, and the sections were prepared. After deparaffinization, the antigen in the section was activated by immersion in 10 mM target retrieval solution (Dako) in a microwave oven operating for 2 min. The sections were blocked in TBS with 10% FBS for 10 min and incubated with the anti-MMP3 C-terminal antibody (1:100) in TBS containing 3% BSA at 4°C overnight. Afterwards, the sections were incubated with anti-rabbit IgG tetramethyl rhodamine isothiocyanate conjugate (1:100) for 1 h in TBS containing 3% BSA and subsequently were incubated with 10 nM Sytox green nucleic acid stain (Molecular Probes, Invitrogen) in TBS for 1 h. Fluorescent medium (Dako) was used for mounting. The sections were observed by confocal laser scanning microscopy.

To clarify if MMP3 could modulate the CCN2 promoter activity, we cotransfected several cell lines with an MMP3 expression vector (p3xFlagMMP3-myc; schemed in Fig. Fig.3A)3A) and CCN2 promoter reporter constructs and then quantified the promoter activity by conducting luciferase assays. MMP3 expression by transient transfection was confirmed by immunoblotting (Fig. (Fig.3B).3B). The intracellular overexpression of MMP3 activated the CCN2/CTGF promoter (Fig. 3C, D). TGF-β and intracellular MMP3 synergistically activated the CCN2/CTGF promoter (Fig. (Fig.3C).3C). The CCN2 promoter activity with MMP3 overexpression was 2.2-fold higher in HCS-2/8 cells, 1.8-fold higher in MDA231 cells, and 2.2-fold higher in COS7 cells than in their controls (Fig. (Fig.3D).3D). In contrast, the effect of overexpressed MMP3 was not significant in HeLa or SaOS-2 cells (Fig. (Fig.3D).3D). It should be noted that the relative CCN2 promoter activity in HCS-2/8 and MDA231 cells was over 30-fold higher than that in SaOS-2 and HeLa cells (data not shown). To further investigate this mechanism, we utilized mutants of the CCN2 promoter (Fig. (Fig.3E).3E). Mutants pDS4 and pDS5, lacking the element between −202 and −88, lost the response to overexpressed MMP3 in HCS-2/8 cells (Fig. (Fig.3F).3F). The response of the CCN2 promoter to overexpressed MMP3 was diminished by the mutagenesis of TRENDIC but not by that of SBE or BCE1/TbRE in HCS-2/8 cells (Fig. (Fig.3G).3G). Also, in MDA231 cells the TRENDIC mutant of the CCN2 promoter lost the response to overexpressed MMP3 (Fig. (Fig.3H).3H). However, in contrast to the result obtained with HCS-2/8 cells, mutations in BCE1/TbRE deprived the CCN2 promoter of the response to overexpressed MMP3 in MDA231 cells (Fig. (Fig.3H).3H). Based on these results, we concluded that TRENDIC mediated the effect of MMP3 on the activation of the CCN2 promoter in chondrocytic cells. Interestingly, our data also indicated that MMP3 could enhance the CCN2 promoter activity in collaboration with another enhancer element, BCE1/TbRE, in MDA231 cells but not in HCS-2/8 cells.

Next, to clarify the subcellular dynamics of MMP3, we prepared Cy3-labeled rhMMP3 (Cy3-MMP3), added it to the medium of HCS-2/8 cells in culture, and observed its behavior with a confocal laser scanning microscope. The Cy3-MMP3 signals emanated from the cellular membrane or cytoplasm between 5 and 60 min after the addition of Cy3-MMP3 to the cell culture (Fig. (Fig.6A,6A, images b to f, i, and j). Of note, Cy3-MMP3 signals also were observed in the cell nuclei between 15 and 30 min after the addition (Fig. (Fig.6,6, images f to i). The Cy3-MMP3 signal was barely detected at 60 min after the addition, indicating the possibility of degradation or resecretion. The Cy3-MMP3 localization in the Cy3-MMP3-positive cell population was quantitatively analyzed (Fig. (Fig.6B).6B). The Cy3-MMP3 was taken up into the cells within 5 min after the addition, showing dominant localization in the cytoplasm, which was followed by the translocation into the nucleus between 15 and 60 min after the addition. Additionally, to demonstrate that MMP3-Cy3 was certainly in the nucleus, a sequential horizontal view of the MMP3-Cy3 signal and DNA in a cell was presented (Fig. (Fig.6C).6C). These observations clarified that extracellular MMP3 is taken up into the HCS-2/8 cells and subsequently translocated into the nucleus.

In order to demonstrate that nuclear translocated MMP3 trans activates the CCN2/CTGF promoter, leptomycin B (LMB) was employed. LMB is an antibiotic with membrane permeability, and it has been known to inhibit the nuclear export function of CRM1/exportin 1 by directly binding to its cysteine residue. We hypothesized that the nuclear export of MMP3 could be CRM1 dependent, and thus LMB could cause the nuclear accumulation of MMP3 and the enhancement of the activation of the CCN2/CTGF promoter by nuclear MMP3. Exactly as we predicted, LMB enhanced the trans-activation effect of MMP3 on the CCN2/CTGF promoter (Fig. (Fig.6D).6D). The increase in the CCN2 promoter activity without MMP3 overexpression may represent the effect of LMB on endogenous MMP3. These results represent that nuclear-cytoplasmic trafficking is crucial in the trans activation of the CCN2/CTGF promoter by MMP3.

To investigate the relationship between subcellular MMP3 localization and the pathophysiology of articular cartilage in animals, we immunohistochemically examined MMP3 in normal and osteoarthritic articular cartilages with a confocal laser microscope. MMP3 was immunopositive in the normal articular cartilage of 2-month-old mice (Fig. (Fig.9A,9A, row a), while control IgG brought no significant signal. The colocalization of the MMP3 signals with DNA was very evident at high-power magnification (Fig. (Fig.9A,9A, row b). In a rat osteoarthritic cartilage, the articular and semilunar chondrocytes were positively stained by the MMP3 antibody (Fig. (Fig.9B).9B). We also observed the colocalization of the MMP3 staining with DNA in articular chondrocytes at high-power magnification (Fig. (Fig.9B,9B, rows e and f). MMP3 in the cytoplasm also was observed in some osteoarthritic articular chondrocytes (Fig. (Fig.9B,9B, rows e and f; yellow in merged view). It should be noted that the fibrochondrogenic cells, in which CCN2 would be expressed and involved in the tissue remodeling, showed MMP3 existed alongside DNA (Fig. (Fig.9B,9B, row g; yellow in merged view). The data that MMP3 is localized in the nuclei of normal developing and osteoarthritic articular chondrocytes in animals suggest a role of the nuclear MMP3 in the development and regeneration of articular cartilage.