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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.

Methods

OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1β and/or TNFα. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1β–stimulated OUMS-27 cells was investigated.

Results

IL-1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1β–stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up-regulation in OUMS-27 cells.

Conclusion

ADAMTS9 is an IL-1β– and TNFα-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.

Articular cartilage is a highly specialized tissue that covers the bony surfaces of the synovial joints, allowing smooth lubrication and providing resistance to joint forces. Cartilage matrix is synthesized and maintained by chondrocytes, and some of its constituents are collagen types II, IX, and XI, aggrecan, perlecan, cartilage oligomeric matrix protein, and others. Aggrecan, a large aggregating proteoglycan, forms a macromolecular complex with hyaluronan and link protein. It swells within the interstices of the collagen framework and provides compressibility to cartilage (1, 2).

Proteolytic degradation of articular cartilage is a key feature of arthritic joint destruction. The loss of aggrecan is considered to be a critical early event in cartilage destruction, occurring initially at the joint surface and progressing to the deeper zones. This step is followed by degradation of collagen fibrils and mechanical failure of the tissue (2). The degradation of cartilage is mediated by a number of different proteases, including neutral endopeptidases of the metalloproteinase superfamily of enzymes. Members of 2 metalloproteinase families, matrix metalloproteinase (MMP) and ADAMTS, have been implicated in cartilage matrix destruction (1).

The ADAMTS family contains 19 individual gene products (3). Certain members of the ADAMTS family (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15), called aggrecanases, can proteolytically process aggrecan within the interglobular domain separating its globular G1 and G2 domains at a specific Glu373–Ala374 bond (3–5) or at 1 or more sites within the more C-terminal glycosaminoglycan (GAG)–bearing region (6). Proteolytic liberation of the GAG-bearing regions reduces the load-bearing properties of articular cartilage and may accompany or initiate a series of cellular responses that culminate in loss of joint cartilage. These proteases are believed to be active in both inflammatory arthritis and osteoarthritis.

Aggrecanase activity was first detected in bovine articular cartilage treated with interleukin-1β (IL-1β), but it is also enhanced by tumor necrosis factor α (TNFα) or retinoic acid (7, 8). These data support the hypothesis that aggrecanases are active early in the disease process of arthritis or during acute inflammatory episodes. However, the exact enzyme(s) responsible for cartilage aggrecan degradation, both during active inflammation and as arthritis progresses, are still unclear (9). ADAMTS-4 (aggrecanase 1), ADAMTS-5 (aggrecanase 2), and subsequently, ADAMTS-1 were the first proteases to which significant aggrecanase activity was attributed (4–6), although their specific importance in the context of arthritis is not yet fully established. Like ADAMTS-4 and ADAMTS-1, ADAMTS-9 and, more recently, ADAMTS-8 and ADAMTS-15 were shown to be aggrecanases (10–12). In gene profiling studies, ADAMTS-9 was expressed in the setting of osteoarthritis (9, 13). Findings of these experimental studies, observations from phylogeny of the ADAMTS proteases (3, 11), and the absence of aggrecanase activity in numerous other ADAMTS proteases suggest that the enzymes examined in this study include the complete set of ADAMTS aggrecanases. To further investigate the significance of each of these, it would be important to study their expression and induction by cytokines in chondrocytes.

Because normal articular cartilage is relatively acellular and difficult to obtain, in these studies we instead used OUMS-27 cells, a recently established cell line with chondrocytic properties (14). The effects of 2 major proinflammatory cytokines, IL-1β and TNFα, were investigated to determine how and which aggrecanase may play a role in inflammatory disease of cartilage

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Reagents.

Recombinant human IL-1β and TNFα were purchased from R&D Systems (Minneapolis, MN). The cytokines were stored at −80°C and diluted in culture medium immediately before being used. Antibodies to phosphorylated MAPKs were purchased from Cell Signaling Technology (Beverly, MA). The p44/42 MAPK inhibitor PD98059 and the p38 MAPK inhibitor SB203580 were purchased from Calbiochem (San Diego, CA) and dissolved in DMSO. All other chemicals and biochemicals were from Sigma (St. Louis, MO).

Cell culture.

OUMS-27 cells were a kind gift from Dr. T. Kunisada (Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan). OUMS-27 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air. The cells were subcultured at split ratios of 1:2 to 1:4 using trypsin plus EDTA every 7–10 days. The medium was changed every 3 days. Cells were used at passages 7–14 for all experiments. For most experiments, 5 × 105 cells were plated in 60-mm dishes and transferred to serum-free DMEM for 24 hours and then exposed to the different cytokines and inhibitors at the concentrations and times indicated.

Human articular chondrocytes (Cryo NHAC-kn) were purchased from Sanko Junyaku (Tokyo, Japan), and cultures were maintained according to the manufacturer's protocol. Chondrocytes between passages 3 and 7 were used for the analysis. Human skin fibroblasts (HSFs) were kindly provided by Dr. S. Hattori (Nippi Research Institute of Biomatrix, Tokyo, Japan). HSFs were cultured as previously described (15).

Generation of ADAMTS-9 antibody.

A synthetic peptide, CQHPFQNEDYRPRSASPSRTH, derived from the human ADAMTS-9 amino acid sequence (13), was synthesized at the National Institutes of Health–supported Lerner Research Institute Biotechnology core, conjugated to keyhole limpet hemocyanin, and used as an immunogen in rabbits to generate polyclonal antisera (Alpha Diagnostics International, San Antonio, TX). The antiserum was affinity-purified against the immobilized peptide immunogen. It was tested by Western blot analysis of HEK293F cells stably transfected with full-length ADAMTS-9. Lysate and conditioned medium of stably transfected cells, but not nontransfected cells, demonstrated a reactive band of ∼180–200 kd on denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which is compatible with the predicted molecular mass of ADAMTS-9. Other bands of 100 kd and 75 kd were identified in nontransfected 293 cells and are believed to represent cross-reactivity with proteins other than ADAMTS-9.

Cytokine stimulation and protein kinase inhibitor assay.

All cells were first incubated in 4 ml of medium containing 10% FBS. After 72 hours, the medium was changed to serum-free DMEM, and the cells were incubated for another 24 hours. The cells were then exposed to various concentrations of IL-1β (10–100 ng/ml) and/or TNFα (5–50 ng/ml) in phosphate buffered saline (PBS) or PBS containing 0.1% bovine serum albumin as a control (n = 4 each), according to a previously described protocol (16, 17).

For kinase assays, cells were cultured on 60-mm dishes and serum-starved for 24 hours before being stimulated. OUMS-27 cells were pretreated with 10–100 μM PD98059, a specific inhibitor of p44/42 kinase, 5–50 μM SB203580, a specific inhibitor of p38 kinase, or DMSO alone (as a vehicle control) for 30 minutes and subsequently incubated with IL-1β (10 ng/ml).

RNA preparation and reverse transcription–polymerase chain reaction (RT-PCR).

Following stimulation, the cells were washed once with PBS, and total RNA was extracted using TRIzol according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Residual DNA was removed by treatment with 5 units of DNase I (Roche Diagnostics, Lewes, UK) at room temperature for 15 minutes followed by inactivation at 65°C for 10 minutes. Two micrograms of total RNA was reverse transcribed to complementary DNA (cDNA) with random primers according to the manufacturer's protocol (Toyobo, Osaka, Japan). Primers for PCR were designed to amplify 90–700–bp fragments for each gene (ADAMTS1, ADAMTS4, ADAMTS5, ADAMTS8, ADAMTS9, ADAMTS15, COL2A1, COL9A1, aggrecan, and β-actin) (Table 1). RT-PCR was performed for 35 cycles (25 cycles for β-actin) of incubation at 94°C for 30 seconds, 60°C (57°C for COL2A1 and COL9A1) for 30 seconds, and 72°C for 30 seconds, with the final incubation at 72°C for 7 minutes.

Table 1. Primers used in the polymerase chain reaction analysis
GenePrimer
ADAMTS1Forward: 5′-GGACAGGTGCAAGCTCATCTG-3′
 Reverse: 5′-TCTACAACCTTGGGCTGCAAA-3′
ADAMTS4Forward: 5′-AGGCACTGGGCTACTACTAT-3′
 Reverse: 5′-GGGATAGTGACCACATTGTT-3′
ADAMTS5Forward: 5′-TATGACAAGTGCGGAGTATG-3′
 Reverse: 5′-TTCAGGGCTAAATAGGCAGT-3′
ADAMTS8Forward: 5′-ACCATGTGGTGGACTCGCCT-3′
 Reverse: 5′-GTTCCCATCGTTCTGCACAC-3′
ADAMTS9Forward: 5′-GGACAAGCGAAGGACATCC-3′
 Reverse: 5′-ATCCATCCATAATGGCTTCC-3′
ADAMTS15Forward: 5′-GTGGGGGAGACAATAAGAGC-3′
 Reverse: 5′-GGTACTTGCCTTGGCTGTTC-3′
AggrecanForward: 5′-AAACCACCTCTGCATTCCAC-3′
 Reverse: 5′-CCTCTGTCTCCTTGCAGGTC-3′
COL2A1Forward: 5′-AACTGGCAAGCAAGGAGACA-3′
 Reverse: 5′-AGTTTCAGGTCTCTGCAGGT-3′
COL9A1Forward: 5′-GTGTTGCTGGTGAAAAGGGT-3′
 Reverse: 5′-GGGATCCCACTGGTCCTAAT-3′
β-actinForward: 5′-TTCCTGGGCATGGAGTCCT-3′
 Reverse: 5′-AGGAGGAGCAATGATCTTGATC-3′

Quantitative real-time RT-PCR.

Quantitative real-time RT-PCR analysis was performed using a LightCycler Rapid Thermal Cycling system (Roche Diagnostics), according to a previously reported protocol (18, 19). The PCR mixture consisted of 1× SYBR Green PCR Master Mix, which includes DNA polymerase, SYBR Green I dye, dNTPs (including dUTP), PCR buffer, 10 pmoles of forward and reverse primers, and cDNA of samples, in a total volume of 20 μl. Amplification of a housekeeping gene, β-actin, was used for normalizing the efficiency of cDNA synthesis and the amount of RNA applied. To validate the specificity of amplification of ADAMTS and β-actin, we analyzed each PCR product by agarose gel electrophoresis after real-time detection. Each sample was amplified in duplicate or triplicate. Negative controls were performed with samples in which the RNA templates were replaced by nuclease-free water in the reactions. The intraassay and interassay coefficients of variation were <5% and were reasonably small compared with those reported in other studies. Subsequently, the threshold cycle, i.e., the number of cycles at which the amount of the amplified gene of interest reached a fixed threshold, was determined.

Northern blot analysis.

Northern blot analysis was performed as previously described (19, 20). Total RNA was electrophoresed on a 1% agarose gel, blotted onto Hybond-NX nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridized with α32P-dCTP–radiolabeled probes (Amersham Pharmacia Biotech). The mouse aggrecan (NM_007424) cDNA fragment was cloned between the Not I and Eco RI sites of vector pT7T3D and used for Northern blot analysis. The ADAMTS9 probe was a 960-bp fragment of mouse ADAMTS9 cDNA, as previously reported (13). The ADAMTS5 probe used for the Northern blot analysis was amplified by RT-PCR (182 bp) and subcloned into TA cloning vector, as previously reported (21). Hybridization was carried out at 65°C overnight in Church buffer. The membrane was washed and then exposed to x-ray film (Kodak, Tokyo, Japan) with an intensifying screen. The radiolabeled bands were densitometrically quantified using a BAS image analysis system (Fuji Film, Tokyo, Japan).

Protein extraction.

Cells were incubated with or without cytokines in the medium, as previously reported (22, 23). After stimulation, the cells were washed once with PBS and then scraped from the culture dish. Cell pellets were solubilized in 400 μl of cell lysis buffer (Sigma) with a complete protease inhibitor cocktail (Sigma). For phosphorylation analysis, cells were washed with PBS and lysed in buffer containing 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, and complete protease inhibitor cocktail. After incubation in a rotator at 4°C for 15 minutes, the samples were centrifuged, and the supernatants were collected. The protein concentration of the cell extracts was determined by using a protein assay kit (Bio-Rad, Hercules, CA).

Western blot analysis.

Cell lysates (15 μg of total protein/lane) were subjected to SDS-PAGE using a 4–12% gradient gel and then transferred onto nitrocellulose membranes (Advantech, Tokyo, Japan). Membranes were blocked with 5% skim milk and 0.05% Triton X-100 in PBS and then incubated with primary antibody in Triton X-100 overnight. Membranes were washed with Triton X-100 and then incubated with the appropriate horseradish peroxidase–conjugated secondary antibody diluted in blocking buffer. Immunoreactive proteins were detected using the enhanced chemiluminescence system (Amersham Pharmacia Biotech). To examine the regulation of ADAMTS-9 protein expression by IL-1β, chondrosarcoma cells were incubated with medium in either the presence or the absence of IL-1β (10 ng/ml). Protein expression in cell lysates was determined by Western blot analysis.

Statistical analysis.

Data are expressed as the mean ± SD. Statistical comparisons of means were performed by analysis of variance (ANOVA) followed by Student's paired t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Expression of cartilage-specific extracellular matrix (ECM) genes and ADAMTS genes in OUMS-27 cells.

Initial RT-PCR experiments were performed to determine the expression of messenger RNA (mRNA) for cartilage-specific proteoglycan (i.e., aggrecan) and other ECM genes in cultured OUMS-27 cells. The cDNA were prepared from unstimulated cultured OUMS-27 cells and amplified with specific primers for aggrecan, COL2A1, COL9A1, or β-actin (Table 1). The PCR products were separated by electrophoresis on an agarose gel, and specific bands corresponding to each gene product (COL2A1, 621 bp; COL9A1, 159 bp; aggrecan, 501 bp) were observed (Figure 1A).

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Figure 1. A, Expression of cartilage-specific extracellular matrix genes examined by reverse transcription–polymerase chain reaction (PCR) in cultured OUMS-27 cells. The PCR products were electrophoresed on a 1.2% agarose gel. Single specific bands corresponding to each gene product (COL2A1, 621 bp; COL9A1, 159 bp; aggrecan, 501 bp; negative control [−], using H2O as template]) were observed. β-actin served as an internal control for each sample. The 100-bp molecular weight marker is shown in the far left lane. B, The level of aggrecan mRNA was decreased by interleukin-1β (IL-1β) treatment. A single band of ∼7.3 kb corresponding to aggrecan mRNA was observed. Northern blot analysis demonstrated a change in the aggrecan mRNA expression level in cells stimulated with IL-1β (10 ng/ml) at various time points. 28S and 18S ribosomal RNA bands stained with ethidium bromide are shown to indicate RNA loading in each lane. C, Expression of ADAMTS genes in cultured OUMS-27 cells. The PCR products were electrophoresed on a 1.2% agarose gel. Single specific bands corresponding to each gene product except ADAMTS8 (ADAMTS1, 90 bp; ADAMTS4, 241 bp; ADAMTS5, 182 bp; ADAMTS9, 303 bp; ADAMTS15, 183 bp; negative control [−]) were observed. β-actin served as an internal control for each sample. The 100-bp molecular weight marker is shown in the far left lane. Note that ADAMTS9 mRNA was strongly expressed in unstimulated OUMS-27 cells.

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Next we examined whether the expression level of aggrecan mRNA was altered by IL-1β in OUMS-27 cells. As previously observed in bovine and human chondrocytes (16, 24), IL-1β attenuated the expression of the aggrecan gene in OUMS-27 cells in a time-dependent manner (Figure 1B). These data indicate that despite being cancer cells, OUMS-27 cells retain their chondrocytic phenotype to at least a certain degree.

To determine whether the ADAMTS genes were expressed in cultured OUMS-27 cells under basal conditions, we amplified the cDNA from unstimulated OUMS-27 cells by using ADAMTS-specific primers. The PCR products were separated by electrophoresis on an agarose gel, and specific bands corresponding to each gene product (ADAMTS1, 90 bp; ADAMTS4, 241 bp; ADAMTS5, 182 bp; ADAMTS8, 194 bp; ADAMTS9, 303 bp; ADAMTS15, 183 bp) were observed in all instances except for ADAMTS8 (Figure 1C).

Time course of aggrecanase expression after IL-1β stimulation.

OUMS-27 cells were cultured in the presence of IL-1β (10 ng/ml), and total RNA was extracted at 3, 6, 12, 24, and 48 hours. The relative levels of individual aggrecanase gene mRNA expression were determined by quantitative real-time RT-PCR and compared with those in unstimulated cells (n = 3 independent experiments). The polygonal-to-round chondrocytic shape was maintained in OUMS-27 cells during stimulation (Figure 2A). As shown in Figure 2B, ADAMTS1 mRNA expression was not increased, but rather was decreased, by IL-1β stimulation. IL-1β increased the aggrecanase 1 (ADAMTS4) mRNA expression level; the increase was first noted at 6 hours, and it reached a maximum at 12 hours, after which time it declined. The aggrecanase 2 (ADAMTS5) mRNA expression level also reached a peak at 12 hours and decreased at 48 hours. The ADAMTS9 mRNA expression levels peaked at 6 hours and then gradually decreased. At 6 hours, expression of ADAMTS9 was increased 13.3-fold compared with that in unstimulated cells. ANOVA revealed that this elevation was significant (P < 0.01). ADAMTS8 was not expressed in unstimulated OUMS-27 cells (Figure 1C) or IL-1β–stimulated OUMS-27 cells (data not shown).

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Figure 2. A, Maintenance of the polygonal-to-round chondrocytic shape in OUMS-27 cells after stimulation with IL-1β. B, Quantitative real-time reverse transcription–PCR (RT-PCR) analysis of the expression of ADAMTS genes (ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS9) in IL-1β–stimulated OUMS-27 cells. OUMS-27 cells were treated for 0–48 hours with IL-1β (10 ng/ml). Values are the mean and SD of 3 independent experiments. Note that the increase in the expression levels of ADAMTS9 mRNA is greater than that of other ADAMTS members in IL-1β–stimulated samples. C, Induction of ADAMTS9 mRNA by IL-1β in chondrocytes. Human chondrocytes were cultured in the presence of IL-1β for 0–24 hours, and the expression level of ADAMTS9 mRNA was compared with that in the unstimulated control. Relative expression levels of ADAMTS9 mRNA were determined by quantitative real-time RT-PCR. Values are mean and SD of 3 independent experiments. Note that the time-dependent induction pattern of ADAMTS9 mRNA expression by IL-1β in chondrocytes was similar to that in OUMS-27 cells. ∗ = P < 0.05 versus unstimulated cells. See Figure 1 for other definitions.

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We then examined whether ADAMTS9 is induced in articular chondrocytes by IL-1β stimulation. The rounded polygonal shape of chondrocytes was maintained in monolayer culture during the stimulation (results not shown). As shown in Figure 2C, ADAMTS9 was significantly induced by IL-1β in chondrocytes, with peak expression occurring at 6 hours. The induction of ADAMTS9 mRNA by IL-1β in chondrocytes occurred in a time-dependent manner, similar to that in OUMS-27 cells.

ADAMTS9 expression in OUMS-27 cells.

Because ADAMTS9 was the aggrecanase gene most highly expressed without stimulation and most highly inducible by IL-1β in OUMS-27 cells, we further characterized expression of ADAMTS9 mRNA as well as that of ADAMTS4 mRNA and ADAMTS5 mRNA, by Northern blot analysis. OUMS-27 cells were stimulated with IL-1β (10 ng/ml) for 1, 3, 6, 12, 24, 48, or 72 hours and compared with unstimulated controls (n = 3 independent experiments, respectively). ADAMTS9 mRNA expression was first noted at 3 hours and then further increased and reached a maximum at 6 hours; this pattern of expression was similar to that in human articular chondrocytes. Expression of ADAMTS9 then decreased toward the baseline level (Figure 3A). The expression level of ADAMTS4 was very low, and the bands were not detected by Northern blot analysis (results not shown). The positive band for ADAMTS5 was faintly detectable at 6 hours and 12 hours, but expression levels were much weaker than those of ADAMTS9 (Figure 3A).

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Figure 3. A, Top, Northern blot analysis of ADAMTS9 mRNA expression in IL-1β–stimulated OUMS-27 cells. A single band of ADAMTS9 mRNA was detected at the expected size. Middle, Northern blot analysis of ADAMTS5 mRNA expression in IL-1β–stimulated OUMS-27 cells. A single faint band of ADAMTS5 mRNA was detected at the expected size. Bottom, Signals for 28S ribosomal RNA served as an internal control for each sample. B, Western blot analysis for ADAMTS9 in IL-1β–stimulated OUMS-27 cells. Cells were treated for 0–48 hours in serum-free Dulbecco's modified Eagle's medium in the absence (0) or presence of recombinant human IL-1β at 10 ng/ml. Arrow indicates ADAMTS-9 protein (∼180 kd). The bands detected at approximately 75 kd and 100 kd are considered to be nonspecific. Note that expression of ADAMTS-9 protein also peaked at 6 hours after IL-1β stimulation. Western blotting of β-actin was used to evaluate protein loading. C, Representative results of real-time PCR analysis of the dose-dependence of induction of ADAMTS9 mRNA by IL-1β. OUMS-27 cells were cultured in the presence of various concentrations of IL-1β (0, 10, 50, or 100 ng/ml) for 6 hours, and the expression level of ADAMTS9 mRNA was compared with that in the unstimulated control. Relative expression levels of ADAMTS9 mRNA were determined by quantitative real-time reverse transcription–PCR (RT-PCR). D, Representative results of real-time PCR analysis of the induction of ADAMTS9 mRNA by tumor necrosis factor α (TNFα) stimulation. OUMS-27 cells were cultured in the presence of TNFα (5 or 50 ng/ml) for 6 hours, and the expression level of ADAMTS9 mRNA was compared with that in the unstimulated control. Relative expression levels of ADAMTS9 mRNA were determined by quantitative real-time RT-PCR. See Figure 1 for other definitions.

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We next examined ADAMTS-9 protein production in IL-1β–stimulated OUMS-27 cells. Protein was extracted from cells with or without IL-1β stimulation (n = 4 independent experiments, respectively), and Western blot analysis was performed using anti–ADAMTS-9 polyclonal antibody. A strong band of ∼180–200 kd, which likely represents an active form of ADAMTS-9, was clearly observed, as expected (Figure 3B). We were not able to detect ADAMTS-9 protein in the OUMS-27 cell culture medium, suggesting that the secreted enzyme may be cell-anchored, as previously described.

Cytokine induction of ADAMTS9 gene expression in OUMS-27 cells.

Next, the dose-response of ADAMTS9 mRNA to IL-1β and TNFα stimulation was examined. OUMS-27 cells were cultured in the presence of IL-1β (0–100 ng/ml) or TNFα (0–50 ng/ml) (n = 3 independent experiments, respectively). IL-1β caused dose-dependent induction of ADAMTS9 mRNA (Figure 3C). TNFα alone induced ADAMTS9 mRNA expression as well, as previously observed in retinal pigment epithelium cells (25), although it did not cause a linear dose-dependent induction of ADAMTS9 in OUMS-27 cells (Figure 3D).

Synergistic induction of ADAMTS9 mRNA expression in chondrosarcoma cells by the combination of IL-1β and TNFα.

Synergistic induction of ADAMTS in chondrosarcoma cells treated with the combination of IL-1β (10 ng/ml) and TNFα (10 ng/ml) was examined at the 6-hour time point (Figure 4A). At 6 hours, the expression of ADAMTS9 mRNA was markedly up-regulated (33-fold) by the combination of IL-1β and TNFα. We also compared IL-1β–induced expression of the ADAMTS9 gene in chondrocytes and fibroblasts (Figure 4B). ADAMTS9 was induced in both chondrocytes and HSF cells. Interestingly, augmentation of the expression level of ADAMTS9 mRNA was greater in chondrocytes than in HSF cells. Furthermore, the induction pattern of ADAMTS9 by IL-1β and/or TNFα was different in these 2 cell lines. That is, in chondrocytes, the expression level of ADAMTS9 mRNA was augmented more by IL-1β than by TNFα, and the opposite was the case in HSF cells. In addition, the synergistic effect of the combination of IL-1β and TNFα was greater in chondrocytes than in HSF cells (Figure 4B). In contrast, ADAMTS4 mRNA expression appeared to be only moderately up-regulated by IL-1β and TNFα, and no induction of mRNA for ADAMTS5 was observed at 6 hours (data not shown).

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Figure 4. A, Synergistic induction of ADAMTS9 mRNA expression in OUMS-27 cells by the combination of IL-1β and tumor necrosis factor α (TNFα). Medium was replaced with serum-free Dulbecco's modified Eagle's medium supplemented with recombinant human IL-1β (10 ng/ml), TNFα (10 ng/ml), or both cytokines for 6 hours. Relative expression levels of ADAMTS9 mRNA were compared with those of unstimulated control cells by quantitative real-time reverse transcription–PCR (RT-PCR) analysis. Values are the mean and SD of 3 independent experiments. B, Synergistic induction of ADAMTS9 mRNA expression in chondrocytes by the combination of IL-1β and TNFα. Human chondrocytes or human skin fibroblasts (HSFs) were cultured in the presence of recombinant human IL-1β (10 ng/ml), TNFα (10 ng/ml), or both cytokines for 6 hours, and ADAMTS9 mRNA expression levels were compared. Relative expression levels of ADAMTS9 mRNA were determined by quantitative real-time RT-PCR. Values are the mean and SD of 3 independent experiments. See Figure 1 for other definitions.

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IL-1β–stimulated phosphorylation of MAPKs in OUMS-27 cells.

In chondrocytes, activation of the signaling pathways of MAPKs, including ERK and p38, by IL-1β stimulation has been reported to occur (26, 27). SB203580, a specific inhibitor of p38, almost completely blocked the induction of MMP-13 by IL-1β stimulation (26), suggesting its crucial role for induction of the MMP-13 gene. However, these pathways have not been examined in chondrosarcoma cells, and the relationship between these signaling pathways and ADAMTS induction has not been studied. To investigate the molecular mechanism of the up-regulation of ADAMTS9 by IL-1β, OUMS-27 cells were stimulated with IL-1β for 10–120 minutes, and the time course of phosphorylation of MAPKs, p38, and p44/42 was examined. IL-1β immediately (at 10 minutes) increased the phosphorylation of p38, and this increase was terminated at 60 minutes (Figure 5A). Phosphorylation of p44/42 peaked at 10 minutes and then declined to the control level at 30 minutes (Figure 5A). Thus, IL-1β activates the p38 and p44/42 pathways in IL-1β–stimulated OUMS–27 cells.

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Figure 5. A, Time-dependent phosphorylation of p38 and p44/42 MAP kinases in OUMS-27 cells after stimulation with IL-1β. Total protein extracts were subjected to polyacrylamide gel electrophoresis, and Western blot analysis was performed with phosphorylated p38 (P-p38) and phosphorylated p44/42 (P-p44/42) antibodies as well as with antibodies to total with phosphorylated p38 and phosphorylated p44/42. B, SB203580, a p38 MAPK inhibitor, attenuated IL-1β–induced ADAMTS9 mRNA expression. OUMS-27 cells cultured in serum-free medium were pretreated with SB203580 for 30 minutes and then stimulated with IL-1β for 6 hours. The expression levels of ADAMTS9 mRNA were measured. C, PD98059, a p44/p42 MAPK inhibitor, attenuated the IL-1β–induced ADAMTS9 mRNA expression. OUMS-27 cells cultured in serum-free medium were pretreated with SB203580 for 30 minutes and then stimulated with IL-1β for 6 hours. In B and C, results shown are representative of 3 independent experiments using real-time PCR analysis. See Figure 1 for other definitions.

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To investigate the role of the p38 pathway in IL-1β–induced ADAMTS9 expression, OUMS-27 cells were pretreated with SB203580 or PD98059. The effects of protein kinase inhibitors on ADAMTS9 mRNA expression were measured by quantitative real-time RT-PCR at 6 hours. No obvious cytotoxic effect on the OUMS-27 cells was observed when these protein kinase inhibitors were used during the study. SB203580 dose-dependently, but only partially, reduced induction of ADAMTS9 mRNA (Figure 5B). PD98059 also caused the attenuation of ADAMTS9 gene induction (Figure 5C).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In this study, we sought to identify which ADAMTS aggrecanases could be induced by cytokines representative of some of the complex inflammatory cascades in arthritis. We showed that ADAMTS genes were differentially regulated, and that ADAMTS9 was the most highly induced of the aggrecanase family genes in IL-1β–stimulated chondrosarcoma-derived cells and isolated chondrocytes. Induction of ADAMTS9 by cytokine stimulation was greater in OUMS-27 cells and chondrocytes compared with that in fibroblasts.

The difficulty in studying inflammatory responses of cartilage is partly attributable to the lack of available chondrocyte cell lines. OUMS-27 cells were originally isolated from a patient with chondrosarcoma. Although OUMS-27 cells are cancer cells, they maintain the chondrocytic phenotype. For instance, OUMS-27 cells maintained their polygonal-to-round shape, which is the typical morphology of chondrocytes, throughout this study. Furthermore, OUMS-27 cells expressed cartilage-specific proteoglycan (i.e., aggrecan) as well as type II and type IX collagen genes, which are considered to be cartilage specific. Aggrecan gene expression and its attenuation by IL-1β stimulation, which is notably observed in IL-1β–stimulated chondrocytes (24), was observed in OUMS-27 cells as well. However, the fact that OUMS-27 cells express type I and type III collagens at low levels (14) indicates that OUMS-27 cells are not identical to normal chondrocytes. Nevertheless, when the limitation of using chondrocytes for arthritis research is considered, our results do suggest the usefulness of OUMS-27 cells. In addition, the induction pattern of the ADAMTS9 gene by cytokine stimulation was similar (e.g., in kinetics and synergistic effect of IL-1β and TNFα) between OUMS-27 cells and chondrocytes. The observation that OUMS-27 cells, like chondrocytes, clearly exhibited a synergistic effect of IL-1β and TNFα suggests that OUMS-27 cells may be suitable as a surrogate model for analysis of cartilage catabolism.

In unstimulated chondrocytes, RT-PCR analysis and Northern blot analysis revealed that basal expression of ADAMTS9 mRNA was higher than that of ADAMTS4 mRNA and ADAMTS5 mRNA, although no protein was detected. Rapid induction of ADAMTS-9 mRNA and protein in IL-1β–stimulated OUMS-27 cells was also observed, suggesting that this protease may be an important early effector of the inflammatory response. Previous studies demonstrated the synergistic induction of ADAMTS4 and ADAMTS5 genes by a combination of IL-1β and oncostatin M (1). These data represent the first demonstration that ADAMTS9 can be induced by IL-1β or TNFα alone, and further show the synergistic induction by the combination of IL-1β and TNFα, indicating that ADAMTS9 is a cytokine-augmented gene in chondrocytes. Taken together, our data and results of previous studies (1, 28) suggest that aggrecanase genes are synergistically induced by cytokines/growth factors, which is relevant to the complex extracellular milieu in arthritis.

Finally, we investigated the inhibitory effects of MAPK on the expression levels of ADAMTS9 mRNA. SB203580 dose-dependently attenuated the induction of ADAMTS9 mRNA, with ∼50% attenuation at a concentration of 5 μM. PD98059 also attenuated ADAMTS9 mRNA induction. Sylvester et al reported that both SB203580 and PD98059 attenuated induction of the ADAMTS4 gene in IL-17–stimulated bovine chondrocytes (29), and Westra et al reported that RWJ 67657, a p38 MAPK inhibitor, attenuated induction of the ADAMTS4 gene in TNFα- and/or IL-1–stimulated human rheumatoid synovial fibroblasts (30). Our results further extend those observations, but in the context of ADAMTS-9 we conclude that p38 and p44/42 MAPKs play a role in the regulation of some, but not all, responses to IL-1β.

Recently, the transcription factors activator protein 1 (AP-1) and NF-κB were shown to be involved in the induction of MMP genes (27, 28). Granet and colleagues reported that the combination of TNFα and IL-1 enhanced expression and additional recruitment of activated AP-1 and NF-κB (31, 32). The fact that the ADAMTS9 gene was induced synergistically by a combination of IL-1β and TNFα suggests involvement of AP-1 and NF-κB in ADAMTS9 gene regulation. With cytokine stimulation, induction of the ADAMTS9 gene in chondrocytic cells and chondrocytes was greater than that in fibroblasts. These results emphasize that the induction of ADAMTS9 may be related to inflammation in the cartilage.

Because ADAMTS-9 is a recently reported enzyme, there are no previous data on its gene regulation and function in arthritic cartilage and little known information about its basic biochemistry. ADAMTS-9 is the largest human ADAMTS protease and is closely related to ADAMTS-20. Both enzymes have a very complex structure that includes 15 thrombospondin type 1 repeats. These proteases are evolutionarily related to GON-1, a metalloprotease required for cell migration in Caenorhabditis elegans (33). ADAMTS-9 is first processed at consensus furin cleavage sites in the secretory pathway to remove the prodomain and generate a 180–200–kd active form. Subsequent to secretion of this form and presentation at the cell surface, it appears that ADAMTS-9 undergoes additional proteolytic processing at the C-terminus (Koo B-H, Somerville RP, Apte SS: unpublished observations), although the precise cleavage sites and mechanisms have not yet been established. No substrates for ADAMTS-9 other than the proteoglycans versican and aggrecan have been identified.

In conclusion, our results show that expression of the ADAMTS9 gene is strongly induced by cytokines in OUMS-27 cells as well as chondrocytes, suggesting its role in inflammatory arthritides. Future studies will address whether the induced secretion plays a major role in degradation of aggrecan or other secreted cartilage molecules.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Drs. T. Furumatsu, Z. Shen, H. Manabe, K. Yamamoto, I. Naito, and A. Ohtsuka and the members of our department for stimulating discussions and comments, and T. Kunisada and Dr. S. Hattori for providing cells used in these studies. We are also grateful to Ms Tomoko Maeda for technical help and to Bon-Hun Koo for affinity purification of ADAMTS-9 antibody. We thank Dr. F. Er, Dr. M. Gunduz, and Mr. H. Turek for their continuous encouragement in our work.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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