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Abstract

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

Objective

Articular chondrocytes are surrounded by an extracellular pool of fibroblast growth factor 2 (FGF-2). We undertook this study to investigate the possible role of FGF-2 in aggrecan catabolism by aggrecanase in human articular cartilage.

Methods

Aggrecan catabolism was induced by interleukin-1α (IL-1α) in normal human articular cartilage and assessed by measuring the release of glycosaminoglycan (GAG) and aggrecanase-dependent fragments by Western blotting with antibodies against neoepitopes. ADAMTS-4 and ADAMTS-5 messenger RNA (mRNA) expression was measured by quantitative real-time reverse transcriptase–polymerase chain reaction. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and tissue inhibitors of metalloproteinases (TIMPs) 1 and 3 was measured by Western blotting. IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay. Proteoglycan synthesis was monitored by 35S-sulfate incorporation.

Results

IL-1α caused cleavage of aggrecan in cultured human articular cartilage explants, with release of GAG and aggrecan fragments containing ARGS and AGEG neoepitopes. This was inhibited by FGF-2 (1–100 ng/ml). Tumor necrosis factor α and retinoic acid also stimulated release of neoepitope, and this was also suppressed by FGF-2. IL-1α induced ADAMTS-4 and ADAMTS-5 mRNA in primary human chondrocytes, and this was inhibited by FGF-2. IL-1α–induced aggrecan breakdown was inhibited by TIMP-1 or by the N-terminal portion of TIMP-3, although FGF-2 did not affect production of the inhibitors TIMP-1 and TIMP-3 when IL-1α was present. FGF-2 did not prevent IL-1α suppression of proteoglycan synthesis and did not negate its ability to stimulate the production of IL-6, IL-8, and MMPs 1, 3, and 13.

Conclusion

Our findings suggest that FGF-2 may play a chondroprotective role in human articular cartilage by controlling the expression and activity of the aggrecanases ADAMTS-4 and ADAMTS-5.

Loss of articular cartilage is a central feature of both osteoarthritis (OA) and rheumatoid arthritis (RA). Cartilage destruction in both diseases is associated with increased production of proteinases, which may be driven by inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-17. The evidence is strong that cartilage resorption is caused by inflammatory cytokines in RA, since they are known to be produced in the diseased joint, and anticytokine therapy has beneficial effects (1). The nature of the stimulus causing chondrocytes to catabolize their matrix in OA is obscure. There is some evidence that the chondrocytes themselves may express IL-1 (2), but the cause and pathophysiologic significance of this are unknown.

Cartilage is made of soluble polymers trapped in a mesh of insoluble fibers largely comprising type II collagen. There are 2 stages in cartilage resorption. First, there is loss of aggrecan, the major soluble component, and this is followed by loss of collagen. The first step is potentially reversible, since mature chondrocytes can resynthesize the aggrecan. The second step is largely irreversible because the chondrocytes of mature articular cartilage are unable to restore the original fibrous network (3).

Aggrecan is a proteoglycan that forms very large aggregates by binding to hyaluronic acid. It is heavily substituted with sulfated glycosaminoglycan (GAG) chains that retain the water that enables cartilage to resist compression. Aggrecan is lost as a result of proteolysis, particularly that due to aggrecanases that cleave the core protein at characteristic sites. The C-terminal fragments passively escape from cartilage, while the N-terminal portions may remain associated with the hyaluronic acid. Following the loss of aggrecan, the collagen fibers are exposed to attack by specific collagenases, such as matrix metalloproteinase 1 (MMP-1) and MMP-13 (4).

The mechanisms by which IL-1 causes cartilage destruction have been investigated in both cell and organ culture. IL-1 increases chondrocyte aggrecanase activity (5–7). The proteinases that carry out the specific cleavages belong to the ADAMTS class of enzymes. ADAMTS-1 (8), ADAMTS-4 (9), ADAMTS-5 (10), ADAMTS-8 (11), and ADAMTS-9 (12) all cleave aggrecan at the specific sites. IL-1 increases expression of ADAMTS-4 and ADAMTS-5 messenger RNA (mRNA) in animal chondrocytes (13, 14), although in human cells, it has been reported to increase only ADAMTS-4 (15–17). ADAMTS-4–knockout mice were found to have no obvious skeletal phenotype; IL-1 caused proteoglycan degradation in their articular cartilage, and the course of a surgically induced model of OA was the same as in normal animals (18). However, the cartilage of ADAMTS-5–knockout mice was resistant to IL-1, and the cartilage proteoglycan was preserved in both RA and OA models (19, 20). Thus, in mice, ADAMTS-5 is strongly implicated as being important in the catabolism of cartilage aggrecan.

In addition to increasing aggrecanase activity, IL-1 also increases expression of several MMPs, including the specific collagenases MMP-1 and MMP-13 (21). MMP-13 has been implicated in cartilage collagenolysis because it preferentially cleaves type II collagen over types I and III collagen (22), and its expression and production are significantly elevated in human OA cartilage (23). While cartilage catabolism has been extensively investigated, relatively little attention has been paid to the possible existence of anticatabolic mechanisms in cartilage. Are there intrinsic factors in articular cartilage that oppose catabolic stimuli such as IL-1? There are old reports that insulin-like growth factor (IGF) (24) and transforming growth factor β (25) can counteract proteoglycan breakdown in animal cartilage stimulated with low-dose IL-1 in vitro. However, the significance of these findings as intrinsic anticatabolic mechanisms in human articular cartilage is unclear. IGF-1 and osteogenic protein 1 (OP-1) have also been reported to inhibit some actions of IL-1 in human cartilage (26, 27). The existence of anticatabolic mechanisms is of potential importance. Their impairment could predispose to tissue degeneration, their augmentation could slow degeneration, and understanding them could suggest new approaches to therapy, particularly for degenerative disease.

We have found that articular chondrocytes are surrounded by an extracellular pool of fibroblast growth factor 2 (FGF-2) (28). This mediates chondrocyte activation when cartilage is loaded (29) and is rapidly released upon cartilage injury (28). FGF-2 induced the synthesis of a number of proteins in porcine chondrocytes, such as tissue inhibitor of metalloproteinases 1 (TIMP-1) and MMPs 1 and 3 (28). These observations prompted us to examine whether FGF-2 affected the response of human articular cartilage to a well-characterized catabolic stimulus such as IL-1. We report that FGF-2 antagonizes the proteoglycan degradation induced by IL-1 or other catabolic stimuli. We also show that FGF-2 inhibits the up-regulation of ADAMTS-4 and ADAMTS-5 induced by IL-1α in human chondrocytes. These findings suggest that perichondral FGF-2 has an anticatabolic chondroprotective function.

MATERIALS AND METHODS

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

Reagents.

Recombinant human FGF-2 (basic FGF) and TNFα were from PeproTech (London, UK). Recombinant IL-1α was prepared in-house (30). Pronase E was from BDH Laboratory Supplies (Poole, UK). Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were from BioWhittaker (Verviers, Belgium). Antibody against ARGS was a kind gift from Prof. Bruce Caterson and Dr. Clare Hughes (Cardiff University, Cardiff, UK). Recombinant ADAMTS-5 (31), TIMPs (32), and aggrecan interglobular domain (IGD) (33) and antibodies against AGEG neoepitope, MMPs, and TIMPs were gifts from Prof. Hideaki Nagase (Imperial College London, London, UK). Antibody against NITEGE neoepitope was a gift from Prof. John Mort (McGill University, Montreal, Quebec, Canada). All other reagents were the best available grade from Sigma-Aldrich (Poole, UK).

Cartilage explant culture.

Human articular cartilage was harvested from the knee joints of patients undergoing surgery for tumors of the lower limb at Royal National Orthopaedic Hospital (Stanmore and London, UK). It was obtained with approval of the local ethics committee and with informed consent of the patients. Full-thickness femoral condylar cartilage without any macroscopic evidence of degeneration was used for all experiments. Cartilage was cut into small cubes (∼3 mm3). Ten pieces of cartilage (∼100 mg wet weight) were put in 1 well of a 24-well plate (n = 3) in 0.5 ml of medium for proteoglycan synthesis experiments, and 3 pieces of cartilage (∼30 mg wet weight) were put in 1 well of a 48-well plate (n = 3) in 0.25 ml of medium for other experiments. Cartilage was rested in serum-free DMEM supplemented with 25 mM HEPES, penicillin/streptomycin, and amphotericin overnight. After washing with serum-free DMEM, cartilage was preincubated with FGF-2 for 30 minutes or with recombinant TIMP for 10 minutes prior to stimulation with IL-1α, TNFα, or retinoic acid (FGF-2 or TIMP remained present) and then further cultured for the indicated periods.

Isolation of primary chondrocytes.

Cartilage was incubated with Pronase E (1 mg/ml/gm of cartilage) for 30 minutes at 37°C, followed by collagenase from Clostridiumhistolyticum (1 mg/ml/gm of cartilage) overnight at 37°C in DMEM containing 10% FCS. The digest was strained then centrifuged at 500g for 5 minutes. Pellets were washed then resuspended in DMEM containing 10% FCS. Cells were plated in 12-well plates at 1.2 million cells per well. Isolated chondrocytes were cultured for 3–5 days in DMEM containing 10% FCS, serum-starved in serum-free DMEM overnight, and then preincubated with FGF-2 for 30 minutes prior to IL-1α stimulation (FGF-2 was still present).

Quantitation of proteoglycan.

The proteoglycan content of the conditioned medium was measured as chondroitin sulfate (CS) with the dimethylmethylene blue (DMMB) assay (34).

Western blotting.

Aliquots of conditioned medium were deglycosylated overnight at 37°C with chondroitinase ABC and keratanase (Seikagaku Kogyo, Tokyo, Japan) in 50 mM Tris HCl (pH 7.5), 50 mM sodium acetate, and 10 mM EDTA. Protein was precipitated with ice-cold acetone, air-dried, and resuspended in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Samples were loaded onto 6% Tris–glycine gel and separated by SDS-PAGE under reducing conditions. For MMPs and TIMPs, samples were loaded onto 10% and 12% Tris–glycine gels, respectively. The protein was electrically transferred onto polyvinylidene difluoride membrane and immunoreacted overnight with the antibodies of interest. After reaction with horseradish peroxidase–linked anti-mouse IgG antibody (against ARGS, MMP-13, and TIMP-3), anti-rabbit IgG antibody (against AGEG), or anti-sheep IgG antibody (against MMPs 1 and 3 and TIMP-1) (1:1,000; Dako, Carpinteria, CA), immunoreactive proteins on the membranes were detected using the ECL Western blot detection system (Amersham Biosciences, Little Chalfont, UK).

RNA isolation and reverse transcriptase–polymerase chain reaction (PCR).

Total RNA from isolated human chondrocytes was extracted with RNeasy mini-columns (Qiagen, Chatsworth, CA). Total RNA (0.5 μg) was reverse-transcribed using Superscript II (Invitrogen, San Diego, CA). Semiquantitative PCR was performed using Ready-to-Go PCR beads (Amersham Biosciences) with the primers for GAPDH (5′-CATGGAGAAGGCTGGGGCTC-3′ [sense] and 5′-ATGAGGTCCACCACCCTGTT-3′ [antisense]) and TIMP-3 (5′-ATGACCCCTTGGCTCGGGCTCATC-3′ [sense] and 5′-TTAGGGGTCTGTGGCATTGATGAT-3′ [antisense]). The amplified DNA was analyzed on 1% (weight/volume) agarose gel and then visualized with ethidium bromide staining. Quantitative PCR was carried out with the TaqMan real-time PCR system (Applied Biosystems, Foster City, CA) using prevalidated primer/probe mixes obtained from the same company. Real-time PCR was performed using a RotorGene 6000 thermocycler (Corbett Research Australia, Mortlake, New South Wales, Australia). Data capture and primary analysis were carried out with RotorGene 6000 software (version 1.7) from the same company. The expression levels of the genes of interest were normalized to those of GAPDH.

Aggrecanase activity assay.

An aliquot of crude conditioned medium (20 μl) was incubated with recombinant IGD substrate (33) for 16 hours at 37°C. The reaction was terminated by boiling the sample in SDS-PAGE sample buffer, and aggrecanase-specific cleavage of the substrate was detected by Western blotting with a rabbit anti-NITEGE antibody.

Enzyme-linked immunosorbent assay (ELISA) for IL-6 and IL-8.

Conditioned medium was harvested after 24 hours of stimulation. The concentration of IL-6 and IL-8 in conditioned medium was measured by ELISA (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. Results were expressed as the mean ± SEM of triplicate cultures.

Proteoglycan synthesis.

To examine the effects of growth factor on proteoglycan synthesis, we measured 35S-sulfate incorporation into GAG (35). Rested human cartilage explants were preincubated for 30 minutes with FGF-2 (20 ng/ml) and then stimulated with IL-1α (10 ng/ml) for another 48 hours in serum-free medium. For the last 6 hours, the medium was replaced with fresh medium (still containing stimuli) which contained 25 μCi/ml of 35S-sulfate (Amersham Biosciences). After culture, cartilage and medium were separately digested at 65°C for 2 hours in 0.25 ml of 0.05M sodium phosphate buffer (pH 6.5) containing 1 mM EDTA, 2 mMN-acetylcysteine, and 25 μg/ml of papain. CS (0.1 ml of 2 mg/ml) was added to medium. GAG was precipitated with 10% cetylpyridinium chloride (CPC) and washed with 3% CPC, then dissolved in 1 ml of formic acid. After mixing in 5 ml of a scintillation mixture, radioactivity was counted in a liquid scintillation counter and normalized to wet weight of cartilage.

Statistical analysis.

Data were compared using one-way analysis of variance with Dunnett's multiple comparison test using Prism 4 software (GraphPad Software, San Diego, CA). 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. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

IL-1α–induced articular aggrecan degradation in human cartilage.

In order to study the effect of FGF-2 on aggrecan degradation, we stimulated human articular cartilage with IL-1α. This causes chondrocyte-dependent cleavage of the protein core of aggrecan. Typically, this occurs at sites where glutamate is N-terminal to alanine or another amino acid residue with a small aliphatic side chain. Such cleavages are made by aggrecanases and are indicated in Figure 1A. The N-terminal fragments are retained in the tissue by their aggregation with hyaluronic acid, while the C-terminal portions escape and accumulate in the culture medium, where they can be detected by Western blotting with antibodies which recognize either the ARGS neoepitope, which arises from aggrecanase cleavage between the G1 and G2 domains, or the AGEG neoepitope, which results from cleavage in the second CS-rich domain (CS-2) (36) (Figure 1A).

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Figure 1. Interleukin-1α (IL-1α) causes aggrecan cleavage in human cartilage. A, The domain organization of human aggrecan is illustrated, showing peptide bonds cleaved by aggrecanases. G1 = G1 domain; IGD = interglobular domain; KS = keratan sulfate–rich region; CS = chondroitin sulfate–rich region. The neoepitope sequences ARGS and AGEG are shown in boldface. B, Human articular cartilage explants from 16-, 23-, and 59-year-old male subjects were left unstimulated (open circles) or were stimulated with IL-1α (10 ng/ml) (solid circles) for 3, 7, 24, or 48 hours. Glycosaminoglycan (GAG) released into conditioned medium was measured by dimethylmethylene blue (DMMB) assay. Data are the mean and SEM of 3 individuals. ∗ = P < 0.05 versus untreated cartilage at each time point. C, Aggrecan fragments released into conditioned medium from a cartilage sample obtained from the 59-year-old male subject were detected by Western blotting using anti-ARGS and anti-AGEG neoepitope antibodies. D, Human articular cartilage explants from a 37-year-old male subject were left untreated (control [Cont]), were freeze-thawed (F/T), or were treated with IL-1α (10 ng/ml) or cycloheximide (CHX; 10 μg/ml) and cultured for 48 hours. GAG release was measured by DMMB assay. Data are the mean and SEM of triplicate cultures. ∗ = P < 0.05 versus untreated cartilage. E, Aggrecan fragments released into conditioned medium were detected by Western blotting using anti-ARGS and anti-AGEG neoepitope antibodies. Results shown are representative of those from 3 different donors.

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Proteoglycan fragments released can also be measured by the reaction of their GAG chains with the metachromatic dye DMMB. IL-1α caused a time-dependent release of GAG (Figure 1B) and of fragments containing the ARGS and AGEG neoepitopes (Figure 1C). The release was detectable after stimulation for 24 hours. The Western blot of the ARGS neoepitope showed several bands. Judging by their size, the slowest band (∼250 kd) was likely to be the fragment 374ARGS–SELE1545. Multiple faster bands (around ∼120 kd) were possibly fragments arising as a result of preexisting cleavages in the first CS-rich domain (CS-1) by MMPs (37). Since C-terminally cleaved aggrecan accumulates with age (38), human cartilage may have heterogeneous aggrecan with different C-termini. Therefore, fragments of different length are released when the NITEGE373–374ARGS cleavage occurs.

There was a basal GAG release (Figure 1B) and aggrecanase-dependent cleavage (Figure 1C) in untreated cartilage. When chondrocytes were killed by repeated freeze-thawing or treated with cycloheximide to stop protein synthesis, the basal GAG release was not affected (Figure 1D). However, the aggrecanase-dependent cleavage was completely abolished (Figure 1E). These results suggest that much of the basal GAG release is passive, while the aggrecanase-dependent cleavage requires cellular metabolism. Therefore, measuring the ARGS fragment release is a more sensitive assay of aggrecanolysis in human cartilage.

FGF-2 inhibits IL-1α–induced aggrecan cleavage.

When the cartilage explants were stimulated with IL-1α in the presence of FGF-2, there was marked suppression of the release of ARGS neoepitope (Figure 2A). FGF-2 showed a trend toward inhibiting GAG release, but in most cases, this did not achieve statistical significance (data not shown). The suppression was also observed when the cartilage was stimulated with either TNFα or retinoic acid (Figures 2B and C, respectively). Figure 2D summarizes the inhibition of IL-1α–induced release of ARGS neoepitope by FGF-2 for 13 different donors (male donors were ages 10, 17, 42, 45, 51, 56, 62, and 67 years and female donors were ages 16, 31, 42, 47, and 62 years). The mean inhibition by FGF-2 at 100 ng/ml was ∼70%. There was no obvious difference between male and female donors in suppression of the release of the neoepitopes ARGS and AGEG by FGF-2 in response to IL-1α (data not shown).

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Figure 2. Fibroblast growth factor 2 (FGF-2) inhibits aggrecan cleavage in human articular cartilage stimulated with catabolic factors. AC, Human articular cartilage explants were preincubated with (1, 10, or 100 ng/ml) or without FGF-2 for 30 minutes, then were left unstimulated or were stimulated with interleukin-1α (IL-1α; 10 ng/ml) (A), tumor necrosis factor α (TNFα; 10 ng/ml) (B), or retinoic acid (Ret A; 1 μM) (C) for another 24 hours. Aggrecan fragments released into conditioned medium were detected by Western blotting using the anti-ARGS neoepitope antibody. The results were reproducible, and representative blots are shown. D, The intensity of all ARGS-positive bands from 13 different donors was quantified and expressed in relation to IL-1α–treated cartilage (assigned a value of 100%). Bars show the mean. ∗∗∗ = P < 0.001.

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FGF-2 inhibits IL-1α–induced ADAMTS-4 and ADAMTS-5 mRNA expression in primary human articular chondrocytes.

Using quantitative PCR in primary human chondrocytes from 4 different donors, we next determined whether FGF-2 affects the expression of ADAMTS-4 or ADAMTS-5 mRNA. ADAMTS-4 mRNA was induced by IL-1α (Figure 3A), and this was inhibited by FGF-2 in a dose-dependent manner. ADAMTS-5 mRNA induction by IL-1α was weak and transient compared with that of ADAMTS-4. The maximum increase of ADAMTS-5 mRNA by IL-1α was ∼2-fold, and this was also inhibited by FGF-2. We also examined the amount of active aggrecanase in the chondrocyte culture medium to see whether FGF-2 inhibited its production (Figure 3B). Treating the chondrocytes with IL-1α increased the amount of active aggrecanase in the medium. This increase was suppressed if the cells were treated with FGF-2 before and during IL-1α stimulation. These results suggest that the inhibitory effect of FGF-2 on the production of active aggrecanase occurs in isolated chondrocytes as well as in cartilage explants.

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Figure 3. Fibroblast growth factor 2 (FGF-2) inhibits ADAMTS-4 and ADAMTS-5 expression in interleukin-1α (IL-1α)–treated human primary chondrocytes. A, Isolated human primary chondrocytes from 23-, 35-, and 56-year-old male subjects and from a 31-year-old female subject were preincubated with (10 or 100 ng/ml) or without FGF-2 for 30 minutes, then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for 4 or 24 hours. Total RNA was isolated and subjected to quantitative polymerase chain reaction for ADAMTS-4 and ADAMTS-5. The expression of these genes was normalized to that of GAPDH, and IL-1α–treated cells at 4 hours were assigned a value of 100%. Data are the mean and SEM of 4 individuals. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. NS = not significant. B, A 20-μl aliquot of conditioned medium (CM) was incubated with recombinant interglobular domain aggrecan substrate (IGD-sub) at 37°C for 16 hours. Cleavage was quantified by Western blotting with anti-NITEGE antibody. The results were reproducible, and representative blots are shown. rADAMTS-5 = recombinant ADAMTS-5.

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Effect of exogenous TIMPs on IL-1α–induced aggrecan degradation.

We investigated the effect of TIMPs on IL-1α–induced aggrecan degradation because FGF-2 has been reported to increase TIMP expression in cartilage (28, 29). IL-1α induction of ARGS and AGEG neoepitopes was inhibited by TIMP-1 at a concentration of 250 nM, but not by TIMP-2 at the same concentration (Figure 4A). The N-terminal portion of TIMP-3 (at a concentration of 100 nM) was strongly inhibitory, as reported for experiments using porcine and bovine cartilage (32). We further examined the inhibitory activity of the N-terminal portion of TIMP-1, which, like the N-terminal portion of TIMP-3, lacks the C-terminal portion of the molecule. The N-terminal portion of TIMP-1 was also observed to suppress neoepitope release. The inhibitory activity of the N-terminal portion of TIMP-1 was stronger than that of the full-length protein (Figure 4B). Suppression of aggrecan degradation by TIMP-1 was not observed in porcine cartilage explants (data not shown). These results suggest that in human cartilage, IL-1α induction of aggrecan cleavage could be controlled by TIMP-1 as well as TIMP-3.

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Figure 4. Effect of tissue inhibitor of metalloproteinases (TIMP) on interleukin-1α (IL-1α)–induced aggrecan cleavage. A, Human articular cartilage explants from an 8-year-old male subject were preincubated with TIMP-1 or TIMP-2 at a concentration of 100 or 250 nM or with the N-terminal portion of TIMP-3 (N-TIMP-3) at a concentration of 100 nM for 10 minutes, then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for another 24 hours. B, Human articular cartilage explants from a 65-year-old male subject were preincubated with the N-terminal portion of TIMP-1, full-length TIMP-1, or the N-terminal portion of TIMP-3 (500 nM) for 10 minutes and then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for another 24 hours. Aggrecan fragments released into conditioned medium were detected by Western blotting using anti-ARGS and anti-AGEG neoepitope antibodies.

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Effect of FGF-2 on the regulation of TIMPs 1 and 3 in human articular cartilage.

The effect of FGF-2 on TIMP expression was examined. TIMP-1 was constitutively produced by untreated cartilage and was increased ∼2-fold by FGF-2, whereas IL-1α alone had no effect. When the cartilage was treated with FGF-2 in the presence of IL-1α, the TIMP-1 level did not increase (Figure 5A). Regulation of TIMP-3 was also examined. TIMP-3 protein strongly associates with extracellular matrix; therefore, heparin was added for the duration of the culture to trap it in the medium. The amount of TIMP-3 that accumulated in the medium was slightly suppressed by IL-1α, but FGF-2 did not affect this (Figure 5B). TIMP-3 mRNA was also slightly inhibited by IL-1α, but FGF-2 did not consistently affect this (Figure 5C). These results suggest that FGF-2 inhibition of IL-1α–induced aggrecan degradation is not due to alteration of TIMPs 1 or 3, since they are not augmented by FGF-2 in the presence of IL-1α.

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Figure 5. Regulation of tissue inhibitors of metalloproteinases (TIMPs) 1 and 3 in human articular cartilage. A, Human articular cartilage explants from a 73-year-old male subject and from 16-, 47-, and 51-year-old female subjects were preincubated with or without fibroblast growth factor 2 (FGF-2) for 30 minutes, then were left unstimulated or were stimulated with interleukin-1α (IL-1α; 10 ng/ml) for another 24 hours. An aliquot of conditioned medium was subjected to Western blotting for TIMP-1. Four separate experiments were carried out with cartilage explants from 4 different donors. Bottom, Results are expressed as the mean and SEM intensity of the TIMP-1 band (n = 4 individuals) with untreated cartilage assigned a value of 100%. ∗ = P < 0.05 versus untreated cartilage. Top, A representative blot is shown. B, Human articular cartilage explants from a 45-year-old male subject were preincubated with FGF-2 for 30 minutes with or without 100 μg/ml of heparin, then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for another 24 hours. An aliquot of conditioned medium was subjected to Western blotting for TIMP-3. C, Isolated human primary chondrocytes from a 47-year-old female subject were preincubated with or without FGF-2 for 30 minutes, then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for another 24 hours. Total RNA was isolated and subjected to reverse transcriptase–polymerase chain reaction for TIMP-3 and GAPDH. Results shown are representative of those from 3 different donors.

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FGF-2 does not inhibit other actions of IL-1α.

To check whether the suppressive effect of FGF-2, which occurred on IL-1α–induced aggrecanase, was also apparent on other IL-1α–induced genes, we studied the induction of MMPs 1, 3, and 13. The collagenases MMP-1 and MMP-13 are implicated in catabolism of type II collagen fibers following loss of aggrecan from the cartilage (4). IL-1α induced MMPs 1, 3, and 13, and this was unaffected by FGF-2 (Figure 6A).

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Figure 6. Effect of fibroblast growth factor 2 (FGF-2) on interleukin-1α (IL-1α) induction of pro–matrix metalloproteinases (proMMPs) 1, 3, and 13 and proteoglycan synthesis in human articular cartilage. A, Human articular cartilage explants from a 42-year-old male subject were preincubated with or without FGF-2 for 30 minutes, then were left unstimulated or were stimulated with IL-1α (10 ng/ml) for another 24 hours. An aliquot of conditioned medium was subjected to Western blotting for MMPs 1, 3, and 13. Three independent experiments were carried out with cartilage explants from 3 different donors. The results were reproducible, and representative data are shown. B, Rested human cartilage explants from a 57-year-old male subject were preincubated for 30 minutes with FGF-2 (20 ng/ml), then stimulated with IL-1α (10 ng/ml) for another 48 hours in serum-free medium. For the last 6 hours, the medium was replaced with fresh medium (still containing stimuli) that contained 35S-sulfate. Radioactive glycosaminoglycan incorporated was counted in a liquid scintillation counter and normalized to wet weight of cartilage. Results are the mean and SEM of triplicate cultures. Two independent experiments were carried out with cartilage explants from 2 different donors. The results were reproducible, and representative data are shown. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. NS = not significant.

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The fact that FGF-2 did not suppress IL-1α induction of MMPs 1, 3, and 13 suggested that its effect on aggrecan catabolism was not due to its acting as an inhibitor of IL-1α. To investigate this further, we measured the effect of FGF-2 on cytokine production induced by IL-1α. IL-6 and IL-8 production by cartilage explants was increased by IL-1α from 2.4 ± 0.3 pg/ml to 184.4 ± 38.0 pg/ml (P < 0.05) and from 7.1 ± 1.8 pg/ml to 112.1 ± 6.92 pg/ml (P < 0.01), respectively, during 24-hour culture. Production of neither cytokine was significantly altered by the presence of 10 ng/ml of FGF-2 (207.8 ± 31.53 pg/ml for IL-6 [P = 0.65] and 103.6 ± 11.2 pg/ml for IL-8 [P = 0.68]).

The effect of FGF-2 on proteoglycan synthesis by cartilage explants was examined by measuring sulfate incorporation. As expected, IL-1α strongly inhibited proteoglycan synthesis (Figure 6B), but FGF-2 did not reverse this.

DISCUSSION

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

Our finding that IL-1α induces both ADAMTS-4 and ADAMTS-5 in human articular chondrocytes is in contrast to several other reports, although IL-1α does up-regulate both enzymes in animal cartilage (13, 14). Flannery et al (16) found no increases in ADAMTS-1, ADAMTS-2, ADAMTS-3, ADAMTS-4, and ADAMTS-5 mRNA in human articular cartilage stimulated with IL-1 for 4 days. Bau et al (15) found that ADAMTS-4 mRNA was induced in isolated human chondrocytes stimulated for 3 days with IL-1β, whereas ADAMTS-5 was unaffected. Moulharat et al (17) showed variable expression of ADAMTS-4 in explants or isolated chondrocytes in response to stimulation with IL-1β for 1 or 2 days, while ADAMTS-5 expression was constitutive and was not altered. In all of these studies, ADAMTS-5 mRNA was not measured at times earlier than 24 hours after stimulation. We found that IL-1α increased both ADAMTS-4 and ADAMTS-5 mRNA in human articular chondrocytes, but the effect was moderate and transient, especially in the case of ADAMTS-5.

There is still controversy concerning which enzymes are responsible for the degradation of cartilage aggrecan. In mice, ADAMTS-5 is the main aggrecanase because deletion of ADAMTS-5, but not ADAMTS-1 or ADAMTS-4, protects against the catabolic action of IL-1 and against aggrecan loss in the development of OA and inflammatory arthritis (18–20, 39). However, Song et al (40) reported that in human cartilage, knockdown of ADAMTS-4, ADAMTS-5, or both enzymes by small interfering RNA, attenuated the degradation of aggrecan in human cartilage stimulated with a combination of TNFα and oncostatin M (40). This suggested that both ADAMTS-4 and ADAMTS-5 contribute to aggrecanolysis in human tissue. Although we found that the induction of ADAMTS-5 mRNA by IL-1α in human chondrocytes was moderate and transient, it might be important because ADAMTS-5 has at least 1,000-fold higher specific activity on aggrecan than does ADAMTS-4 (31).

Exogenous FGF-2 inhibited IL-1α induction of aggrecanase-dependent aggrecan degradation in human articular cartilage. The suppression was dose dependent and was observed at an FGF-2 concentration as low as 1 ng/ml. FGF-2 did not act as an IL-1α inhibitor because it did not affect IL-1α induction of MMPs 1, 3, and 13 or of IL-6 and IL-8 in human cartilage. It also inhibited aggrecan degradation induced by TNFα or retinoic acid. These findings suggest that the effect of FGF-2 on the action of the inflammatory cytokines is restricted and may be relatively specific to aggrecan degradation. The growth factor does not appear to be having a general antiinflammatory effect. Interestingly, FGF-2 significantly suppressed proteoglycan synthesis, and it is known to inhibit the anabolic activity of IGF-1 and OP-1 in human articular cartilage (26, 27). FGF-2 potentially induces MMP production in cartilage. For example, Im et al (41) reported that FGF-2 stimulates the production of MMP-13 in human articular chondrocytes, and we have reported that MMPs 1 and 3 are induced by FGF-2 in porcine articular chondrocytes (28). Taken together, the evidence suggests that FGF-2 may have multiple functions in cartilage homeostasis; it inhibits aggrecanolysis, but it may promote collagenolysis and reduce aggrecan synthesis.

Chondrocytes are surrounded by an extracellular pool of FGF-2 that is bound to the heparan sulfate chains of perlecan (42). This pool appears to be sequestered but activates the cells when cartilage is compressed by loading. This sequestered pool does not prevent IL-1 from inducing aggrecan breakdown in the cultured cartilage. The fact that adding exogenous FGF-2 activates chondrocytes (e.g., it increased TIMP-1 production) suggests that the pericellular perlecan may be saturated with FGF-2. Interestingly, sequestered endogenous FGF-2 is released and activates chondrocytes when cartilage is injured (28), perhaps causing an anticatabolic effect.

It was surprising that TIMP-1 inhibited IL-1α–induced aggrecan degradation in human cartilage. TIMP-1 has much weaker inhibitory activity against ADAMTS-4 and ADAMTS-5 than does TIMP-3 (43, 44). Some reports describe an inhibitory effect of TIMP-1 on cartilage degradation. For example, Arner et al (45) reported that aggrecanase activity in conditioned medium from IL-1β–stimulated bovine nasal cartilage was inhibited by bovine TIMP-1 with a 50% inhibitory concentration of 210 nM, while Hughes et al (46) found that 255 nM TIMP-1 partially inhibited aggrecanase activity of conditioned medium from IL-1α–stimulated porcine chondrocytes. Bonassar et al (47) reported that recombinant human TIMP-1 significantly inhibited retinoic acid– or IL-1β–induced aggrecan degradation in bovine cartilage. On the other hand, Gendron et al (32) showed that the N-terminal portion of TIMP-1 at 1 μM failed to inhibit aggrecanase activity in either IL-1α–treated bovine nasal cartilage or retinoic acid–treated porcine articular cartilage. We also tested 0.5 μM TIMP-1 and the N-terminal portion of TIMP-1 (which inhibited IL-1α–induced aggrecanase activity in human cartilage) in IL-1α–treated porcine metacarpophalangeal cartilage, and we found that neither inhibited the release of GAG or aggrecan neoepitopes (Sawaji Y, et al: unpublished observations).

One could hypothesize that the inhibitory effect of FGF-2 on IL-1α–induced aggrecan catabolism in human cartilage is due to an increase in TIMP-1 production. However, although FGF-2 alone increased TIMP-1 production 2-fold, this was not seen when IL-1α was present. The expression of TIMP-3, a strong inhibitor of ADAMTS-4 and ADAMTS-5, also appeared not to be affected by FGF-2. Taken together, these results indicate that neither TIMP-1 nor TIMP-3 was likely to account for the inhibition of aggrecan degradation.

How does FGF-2 inhibit aggrecan catabolism in human cartilage? Our quantitative PCR showed that FGF-2 inhibited the IL-1α–mediated induction of both ADAMTS-4 and ADAMTS-5 mRNA expression. However, there was a time lag between the change in mRNA expression of the enzymes and the release of aggrecan fragments. The earliest time at which aggrecan fragments could be detected was 24 hours, while the induction of ADAMTS-4 and ADAMTS-5 mRNA expression by IL-1α occurred much earlier. It is possible that aggrecanase proteins are produced in increased amounts following the increase in their mRNA caused by IL-1α, but that it takes time for the enzymes to be secreted to cleave aggrecan and to reach levels at which neoepitopes are detected in the medium. It is also possible that proteolytic processing, which could alter the activity and matrix-binding affinity of the enzymes, takes time. For example, C-terminal processing of ADAMTS-4 by membrane type 4 MMP is required for the aggrecanase cleavage in IL-1–treated bovine cartilage (48). We were unable to investigate the molecular forms of the proteinases that were produced because of lack of suitable antibodies. The amount of the enzymes was below the level of immunodetection in human cartilage. However, even these very low amounts may be sufficient to cleave aggrecan.

Our conclusion is that FGF-2 is an anticatabolic growth factor in human cartilage that inhibits aggrecanase-dependent aggrecan degradation, but that does not have a general antiinflammatory effect. FGF-2–mediated inhibition of ADAMTS-4 and/or ADAMTS-5 mRNA expression may in part explain the inhibition of IL-1α–induced aggrecanolysis, but posttranscriptional and posttranslational mechanisms cannot be excluded.

AUTHOR CONTRIBUTIONS

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

Dr. Sawaji had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Sawaji, Hynes, Saklatvala.

Acquisition of data. Sawaji, Hynes.

Analysis and interpretation of data. Sawaji, Saklatvala.

Manuscript preparation. Sawaji, Vincent, Saklatvala.

Statistical analysis. Sawaji.

Acknowledgements

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

We thank Prof. Bruce Caterson and Clare Hughes of Cardiff University (Cardiff, UK) for the gift of the anti-ARGS monoclonal antibody, and Dr. John Mort of McGill University (Montreal, Quebec, Canada) for the anti-NITEGE antibody. We also thank our colleague, Prof. Hideaki Nagase, for the antiserum to the AGEG neoepitope, for antibodies to human MMPs and TIMP-1, and for recombinant TIMPs and aggrecan substrate as well as for many helpful discussions. We also thank our surgical colleagues, Tim Briggs and Steve Cannon, at Royal National Orthopaedic Hospital (Stanmore and London, UK) for providing specimens.

REFERENCES

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