A Wnt/β-catenin negative feedback loop inhibits interleukin-1–induced matrix metalloproteinase expression in human articular chondrocytes

Authors


Abstract

Objective

The results of recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating, for instance, the expression of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Wnt/β-catenin signaling in interleukin-1β (IL-1β)–induced MMP expression in human chondrocytes.

Methods

Primary cultures of human, murine, and bovine articular chondrocytes as well as human mesenchymal stem cells and mouse embryonic fibroblasts were used in the experiments. Multiple strategies for the activation and inhibition of signaling pathways were utilized. Reporter assays and coimmunoprecipitation were performed to study the interaction between β-catenin and NF-κB.

Results

In contrast to the role of Wnt/β-catenin in animal chondrocytes, in human chondrocytes it was a potent inhibitor of MMP-1, MMP-3, and MMP-13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect was independent of the T cell factor/lymphoid enhancer factor family of transcription factors but rather was attributable to an inhibitory protein–protein interaction between β-catenin and NF-κB. IL-1β indirectly activated β-catenin signaling by inducing canonical Wnt-7B expression and by inhibiting the expression of canonical Wnt antagonists.

Conclusion

Wnt/β-catenin signaling in human chondrocytes had an unexpected anticatabolic role by counteracting NF-κB–mediated MMP expression induced by IL-1β in a negative feedback loop.

Osteoarthritis (OA) is the most common form of arthritis and is a leading cause of mobility-associated disability. OA affects the whole joint and is characterized by progressive degeneration of articular cartilage, mild signs of inflammation, and typical bone changes (1, 2). Although all tissues in the joint are affected by OA, articular chondrocytes are believed to be major mediators of OA pathogenesis by actively promoting cartilage matrix degradation via the expression of matrix metalloproteinases (MMPs) and aggrecanases in response to adverse environmental signals from, e.g., proinflammatory cytokines. In particular, increased production of MMP-1, MMP-3, and MMP-13 by chondrocytes has been associated with cartilage degradation in OA (3–5). Proinflammatory cytokines such as interleukin-1 (IL-1) are potent inducers of cartilage degradation by activating procatabolic NF-κB signaling in chondrocytes, which results in the expression of MMPs in cartilage (6–9).

A proposed role for Wnt/β-catenin in OA is based predominantly on observations in animal models, as follows: 1) in postnatal mouse models, conditional activation of β-catenin signaling in cartilage resulted in increased articular cartilage degeneration by stimulating endochondral ossification and other phenotypes resembling OA (10); 2) activation of Wnt/β-catenin signaling in rabbit and mouse chondrocytes stimulated the expression of cartilage matrix-degrading MMPs (11, 12); 3) in a guinea pig model of spontaneous OA, development of OA was associated with increased β-catenin expression in cartilage (11); and 4) procatabolic factors such as IL-1 that are implicated in OA development induced the expression of various Wnt proteins, resulting in the activation of β-catenin (12, 13). These findings were subsequently corroborated by the observation of increased nuclear β-catenin staining in human OA cartilage compared with control cartilage (10).

In addition, increased expression of the Wnt target gene (WISP1) was observed in both mouse OA models and in human OA cartilage (14). Likewise, differential expression of various Wnt-related genes has been documented in human joint disorders (15–17). For example, canonical Wnt-1 and noncanonical Wnt-5A have been implicated in rheumatoid arthritis (RA) (15, 16), while canonical Wnt-7B is up-regulated in OA cartilage and RA synovium (17). Interestingly, inhibition of β-catenin signaling in articular chondrocytes also caused OA-like cartilage degradation in Col2a1-ICAT–transgenic mice (18). Taken together, multiple lines of evidence have led to the hypothesis that low levels of Wnt/β-catenin signaling are required for maintenance of normal cartilage function, and that dysregulation of this pathway may contribute to the development and progression of OA. Consequently, the Wnt/β-catenin signaling pathway has been identified as a potential therapeutic target for intervention in OA.

To date, functional data on the role of Wnt/β-catenin signaling in human chondrocytes remain scarce. Therefore, in this study, we systematically evaluated the role of canonical Wnt signaling in human chondrocytes compared with animal chondrocytes. Our study revealed an unexpected and remarkable species difference in the regulation of MMP expression by Wnt/β-catenin signaling. In contrast to its procatabolic role in animal models, Wnt/β-catenin signaling in human chondrocytes potently inhibits MMP expression and can effectively counteract procatabolic NF-κB signaling activated by IL-1β in a negative feedback loop.

PATIENTS AND METHODS

Human cartilage samples.

Cartilage samples were obtained from 8 patients (mean ± SD age 62 ± 10 years) with OA and 1 patient (age 67 years) with RA who were undergoing total knee replacement surgery. Knee cartilage was harvested from regions with no macroscopically evident degeneration.

Cell culture and cartilage explant culture.

Primary human and bovine articular chondrocytes were isolated from the cartilage of knee joints, as previously described (8). Human fetal chondrocytes were obtained as previously described (19). Passage 0 or passage 1 chondrocytes were used in all experiments. Human chondrocytes, bovine chondrocytes, HEK 293T cells, and mouse embryonic fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human bone marrow–derived mesenchymal stem cells (MSCs) were isolated from aspirates as described previously (20) and cultured in α-minimum essential medium with 10% FBS and penicillin/streptomycin. For cartilage explant culture, human cartilage was sliced into small cubes and maintained in DMEM with 10% FBS and 1% penicillin/streptomycin. Murine cartilage was isolated from the femoral heads of 2-month-old C57B mice and maintained in DMEM with 10% FBS and 1% penicillin/streptomycin. Normal human articular chondrocytes were derived from a donor (age 45 years) without joint disease (Lonza) and used as controls.

Recombinant proteins and reagents.

Recombinant human Wnt-3A, IL-1β, Dkk-1, and recombinant mouse Wnt-3A (R&D Systems) were used. The glycogen synthase kinase 3 (GSK-3) inhibitor 6-bromoindirubin-3′-oxime (BIO) was obtained from Sigma-Aldrich.

RNA isolation and real-time quantitative polymerase chain reaction (qPCR).

Total RNA was isolated using a NucleoSpin RNA II Kit (Macherey-Nagel). Complementary DNA (cDNA) was synthesized from total RNA, using an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed with a MyiQ Real-Time PCR Detection System (Bio-Rad), using a standard curve–based method (21). GAPDH was used as an internal control. The primer sequences are shown in Supplemental Table 1, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/.

Immunoprecipitation and Western blotting.

Immunoprecipitation was performed using a Dynabeads Co-Immunoprecipitation Kit (Invitrogen). Nuclear and cytoplasmic proteins were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific). Total cell proteins were collected in radioimmunoprecipitation assay buffer (Cell Signaling Technology) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). The antibodies used for Western blotting and coimmunoprecipitation were anti–β-catenin (BD Biosciences), proMMP-1 and proMMP-13 (R&D Systems), TATA box binding protein (Millipore), GAPDH (Sigma-Aldrich), NF-κB p50 (C-19) and NF-κB p65 (A) (Santa Cruz Biotechnology), FLAG (OriGene), T cell factor 4 (TCF-4; Millipore), and lymphoid enhancer factor 1 (LEF-1; Millipore).

MMP activity assay.

Generic MMP activity in human chondrocytes and culture media was measured using a SensoLyte 520 Generic MMP Activity Kit (AnaSpec). MMP activity was normalized to protein concentrations of total cell lysates, as measured using a Pierce BCA Protein Assay Kit (Thermo Scientific).

Plasmid constructs and viral transduction.

Human Wnt-3 and Wnt-7B cDNAs (OriGene) were cloned into a pBOB lentiviral vector (plasmid 12335; Addgene) (22). Short hairpin RNA (shRNA) sequences against human TCF-4 and LEF-1 were cloned into cloning vector pLKO.1-TRC (plasmid 10878; Addgene) (23); pLKO.1 vectors containing a scrambled shRNA (plasmid 18640; Addgene) (24) and an shRNA sequence against human β-catenin (plasmid 18803; Addgene) (25) were used. Lentiviral vectors and packaging vectors were transfected into HEK 293T cells to produce lentiviruses. Lentiviruses were harvested and used to infect chondrocytes in the presence of 6 μg/ml Polybrene (Sigma-Aldrich).

Small interfering RNA (siRNA) transfection.

Mouse embryonic fibroblasts were transfected with ON-TARGETplus SMARTpool siRNA (Thermo Scientific), using X-tremeGENE siRNA Transfection Reagent (Roche).

Reporter assay.

Human chondrocytes were infected with Cignal lentiviruses containing the NF-κB–responsive reporter or a TCF/LEF reporter (SABiosciences) together with lentiviruses constitutively expressing Renilla luciferase (SABiosciences) in the presence of 6 μg/ml Polybrene (Sigma-Aldrich). Luciferase activity was measured using a Dual-Glo Luciferase Assay kit (Promega). The activity of firefly luciferase was normalized to Renilla luciferase activity.

Statistical analysis.

Data were analyzed by Student's 2-tailed t-tests or by one-way analysis of variance and are expressed as the mean ± SD. P values less than 0.05 were considered significant.

RESULTS

Effect of canonical Wnt signaling on MMP messenger RNA (mRNA) expression in human chondrocytes.

To investigate the effect of Wnt/β-catenin signaling on MMP expression, human articular chondrocytes isolated from the knee joints of patients with OA who were undergoing total knee replacement surgery were treated with recombinant human Wnt-3A or the GSK-3 inhibitor BIO. Both treatments activated canonical Wnt signaling in human chondrocytes, as evidenced by an increase in total β-catenin protein levels, nuclear localization of β-catenin, and activation of a canonical Wnt signaling–responsive TCF/LEF reporter (see Supplemental Figures 1A–C, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). The specificity of Wnt-3A for activation of the canonical pathway was demonstrated by coincubation with the extracellular antagonist for canonical Wnt signaling Dkk-1, which blocked Wnt-3A–induced TCF/LEF reporter activity (see Supplemental Figure 1C). Furthermore, treatment with Wnt-3A stimulated chondrocyte proliferation, which is an established effect of canonical Wnt signaling in many cell types (26, 27) (see Supplemental Figure 1D, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/).

We next examined the effect of Wnt/β-catenin signaling on the expression of MMPs and chondrocyte markers. To our surprise, activation of canonical Wnt signaling by either Wnt-3A or BIO significantly decreased the expression of MMP-1, MMP-3, and MMP-13 mRNA in human OA chondrocytes (Figure 1). In agreement with a recent study, the expression of chondrocyte markers COL2A1 and SOX9 was decreased (27) (see Supplemental Figure 1E, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/).

Figure 1.

Effect of Wnt/β-catenin activation on matrix metalloproteinase (MMP) mRNA expression. Human articular chondrocytes from 4 patients with osteoarthritis (OA) were stimulated with 200 ng/ml recombinant human Wnt-3A or 1 μM 6-bromoindirubin-3′-oxime (BIO) or were left untreated (control [CTL]) for 48 hours. Murine cartilage explants (triplicate cultures) were treated with 200 ng/ml recombinant mouse Wnt-3A or 1 μM BIO for 5 days, and bovine chondrocytes (triplicate cultures) were treated with 1 μM BIO for 48 hours. Human cartilage explants were treated for 5 days, and normal human articular chondocytes, human mesenchymal stem cells (MSCs), human rheumatoid arthritis (RA) chondrocytes, and human fetal chondrocytes were treated for 48 hours with 200 ng/ml recombinant human Wnt-3A. Messenger RNA expression was measured by quantitative polymerase chain reaction. Bars show the mean ± SD of triplicate cultures. ∗ = P < 0.05; ∗∗ = P < 0.01 versus control.

We further assessed the effect of Wnt-3A on catabolic gene expression. The decrease in MMP mRNA expression induced by Wnt-3A was dose dependent and gradual; it was first measurable 6 hours after the start of treatment, and maximum inhibition was observed at 72 hours (see Supplemental Figures 1F and G, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). The inhibition could be reversed by Dkk-1, which also efficiently blocked Wnt-3A–induced expression of the established Wnt target gene AXIN2 (see Supplemental Figures 1H and I, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). Notably, blocking of endogenous canonical Wnt signaling by Dkk-1 slightly but significantly increased basal transcription levels of MMP-1 and MMP-13 mRNA (see Supplemental Figure 1H). Taken together, these data suggest that Wnt-3A represses MMP mRNA expression in human chondrocytes via a signaling cascade downstream of the Frizzled/low-density lipoprotein receptor–related protein receptor complex and probably through β-catenin.

In sharp contrast, stimulation of mouse cartilage explants with recombinant mouse Wnt-3A or BIO induced MMP-3 and MMP-13 mRNA expression (Figure 1), which is consistent with previously reported data (12). Similarly, stimulation of bovine chondrocytes with BIO induced MMP-3 and MMP-13 expression (Figure 1). The species difference could not be explained by chondrocyte dedifferentiation, which is an established effect of culturing chondrocytes in monolayer, because direct treatment of human cartilage explants with Wnt-3A also reduced MMP mRNA expression (Figure 1). To exclude the possibility that the observed species difference in the regulation of MMP mRNA expression by canonical Wnt signaling in chondrocytes was attributable to an OA-induced change in cell response, the experiments were repeated using human chondrocytes from a donor without degenerative cartilage disease and from a donor with RA. In cells from these donors, Wnt-3A inhibited MMP-1, MMP-3, and MMP-13 expression (Figure 1). Similar results were obtained using human fetal chondrocytes, excluding an age-related effect (Figure 1). Finally, activation of canonical Wnt signaling in multipotent human MSCs that were able to differentiate into chondrocytes also reduced MMP mRNA expression (Figure 1), suggesting that a decrease in MMP-1, MMP-3, and MMP-13 mRNA expression upon activation of canonical Wnt signaling is a conserved response in various human cell types. Taken together, these data point to a remarkable species difference in the regulation of MMP mRNA expression by Wnt/β-catenin signaling in human and animal chondrocytes.

Effect of Wnt/β-catenin signaling on IL-1β–induced MMP expression and activity in human chondrocytes.

IL-1β is a potent activator of MMP expression in human chondrocytes, and it has been implicated as having a role in cartilage degradation in OA (6–9). To determine the effect of canonical Wnt signaling on IL-1β–induced MMP-1 and MMP-13 mRNA expression, human chondrocytes were costimulated with IL-1β and Wnt-3A or BIO. As expected, IL-1β potently induced expression of MMP-1 and MMP-13 at the mRNA level and proMMP-1 and proMMP-13 at the protein level (Figures 2A and B). Costimulation with Wnt-3A or BIO blocked IL-1β–induced MMP expression, while coincubation with Dkk-1 further increased IL-1β–induced MMP expression (Figure 2A). Both Wnt-3A and BIO inhibited proMMP-1 and proMMP-13 protein expression under basal conditions and after costimulation with IL-1β (Figure 2B). The inhibitory effect of Wnt-3A on MMP-1 and MMP-13 mRNA and protein expression was associated with a decrease in released generic MMP activity in the culture media and in cell extracts. This effect was observed in both basal conditions and after cotreatment with IL-1β (Figure 2C). BIO did not decrease generic MMP activity in basal conditions but inhibited IL-1β–induced MMP activity. These results suggest that activation of Wnt/β-catenin signaling in human chondrocytes negatively regulates the expression and activity of a set of MMP family members both in basal conditions and after stimulation with IL-1β.

Figure 2.

Wnt/β-catenin signaling reduces interleukin-1β (IL-1β)–induced MMP expression and activity. A, Human chondrocytes were treated for 48 hours with 10 ng/ml IL-1β or IL-1β in combination with 200 ng/ml recombinant human Wnt-3A, 1 μM BIO, or 300 ng/ml Dkk-1. MMP-1 and MMP-13 mRNA expression was measured by quantitative polymerase chain reaction. B, Human chondrocytes were treated for 48 hours with 200 ng/ml recombinant human Wnt-3A, 1 μM BIO, or 10 ng/ml IL-1β, and proteins were isolated from cell extracts. ProMMP-1 and proMMP-13 were detected by immunoblotting. C, Culture media (left) and cell lysates (right) of human chondrocytes were collected for generic MMP activity measurements after treatment with 200 ng/ml recombinant human Wnt-3A, 1 μM BIO, 10 ng/ml IL-1β, or combinations of these agents for 72 hours. ∗ = P < 0.05; ∗∗ = P < 0.01. Bars in A and C show the mean ± SD of triplicate cultures (n = 3 donors). See Figure 1 for other definitions.

Cross-talk of Wnt/β-catenin and IL-1β signaling pathways in human chondrocytes.

The potentiating effect of Dkk-1 on MMP-1 and MMP-13 expression in human chondrocytes in both basal and IL-1β–stimulated conditions suggested that the presence of endogenous canonical Wnt family members in human chondrocytes repressed MMP expression. Using a qPCR survey, we identified relatively abundant mRNA expression of noncanonical Wnt-5A and lower expression levels of noncanonical Wnt-4, Wnt-5B, and Wnt-9A, and canonical Wnt-7B (see Supplemental Table 2, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). Wnt-7B was the only canonical Wnt up-regulated by IL-1β. The peak in Wnt-7B mRNA expression occurred 24 hours after IL-1β stimulation, which coincided with the peak in MMP-1 and MMP-13 mRNA expression, after which both MMP-1 and MMP-13 mRNA and Wnt-7B mRNA expression started to decrease (Figure 3A). Interestingly, IL-1β treatment simultaneously down-regulated mRNA expression of several canonical Wnt signaling inhibitors, including Dkk-1, Frizzled-related protein (FRZB), and Wnt inhibitory factor 1 (WIF-1) (Figure 3A). Dkk-1 and WIF-1 mRNA expression started to decrease 4 hours after stimulation and reached the lowest expression levels at 24 hours (Figure 3A). The decrease in FRZB mRNA expression was more gradual (Figure 3A). As a consequence of the opposite effects on the expression of Wnt-7B and its antagonists, β-catenin protein accumulated in both nuclear and cytoplasmic compartments upon stimulation of human chondrocytes with IL-1β (Figure 3B).

Figure 3.

Cross-talk of Wnt/β-catenin and interleukin-1β (IL-1β) pathways. A, Messenger RNA expression of Wnt-7B, MMP-1, and MMP-13, and the Wnt antagonists Dkk-1, Frizzled-related protein (FRZB), and Wnt inhibitory factor 1 (WIF-1) was evaluated at the indicated time points after stimulation with IL-1β by quantitative polymerase chain reaction (qPCR). Data represent the mean fold change in cells from 3 donors relative to untreated cells, which was set at 1. Each experiment was performed in triplicate. At 4 hours, P < 0.05 for MMP-1, MMP-13, Dkk-1, and WIF-1 versus untreated cells; at 72 hours, P < 0.05 for Wnt-7B and MMP-13 versus untreated cells; for comparisons at all other time points, P < 0.01. At 4 hours and 72 hours, FRZB expression was not significantly changed. B, Human chondrocytes were stimulated with 20 ng/ml IL-1β for 48 hours, and nuclear and cytoplasmic β-catenin protein expression was detected by immunoblotting. C, Human Wnt-3 and Wnt-7B were overexpressed using lentiviral transduction in human chondrocytes, and MMP mRNA expression was measured by qPCR. Lentiviral green fluorescent protein (GFP)–transduced cells (Lenti-GFP) were used as control. D, Wnt-7B was knocked down by lentivirus-mediated expression of short hairpin RNA (shRNA) in human chondrocytes using scrambled shRNA (shScr) as control. MMP-1 and MMP-13 mRNA expression was measured 24 hours after stimulation with 10 ng/ml IL-1β and compared with control. Values in C and D are the mean ± SD of triplicate cultures (n = 1 donor). See Figure 1 for other definitions.

Lentiviral overexpression of Wnt-7B as well as Wnt-3, another canonical Wnt not detected in the qPCR survey as control, inhibited MMP-1 and MMP-13 mRNA expression, and this inhibition was similar to that observed following stimulation with Wnt-3A in human chondrocytes (Figure 3C and Supplemental Figure 2A [available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/]). Wnt-7B and Wnt-3 stimulated the expression of the canonical Wnt target gene AXIN2 (see Supplemental Figure 2B, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). Knockdown of Wnt-7B in human chondrocytes increased MMP-1 and MMP-13 mRNA expression in both basal conditions and after stimulation with IL-1β (Figure 3D and Supplemental Figure 2C [available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/]). Taken together, these data suggest that Wnt-7B is a likely candidate for the endogenous canonical Wnt that negatively regulates MMP expression in human chondrocytes. Furthermore, these data point to a role of canonical Wnt signaling as a negative feedback loop controlling MMP expression downstream of IL-1β receptor activation.

No involvement of TCF-4 and LEF-1 in Wnt-mediated repression of MMP expression in human chondrocytes.

Because Wnt/β-catenin showed an inhibitory effect on MMP expression, we next examined whether knockdown of β-catenin by lentiviral shRNA expression could restore MMP expression levels. The knockdown efficiently decreased β-catenin expression (see Supplemental Figure 2D, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/) and strongly elevated MMP expression levels (Figures 4A and B). The inhibitory effect of Wnt-3A was almost completely eliminated by β-catenin knockdown (Figures 4A and B).

Figure 4.

Effects of β-catenin, T cell factor 4 (TCF-4), and lymphoid enhancer factor 1 (LEF-1) knockdown. A–C, Human chondrocytes were transduced with lentiviruses expressing scrambled short hairpin RNA (shScr) and short hairpin RNA against human β-catenin (shCat) (A and B), TCF-4 (shTCF4), and LEF-1 (shLEF1) (C) and treated with 50 ng/ml recombinant human Wnt-3A for 24 hours. Matrix metalloproteinase (MMP) mRNA expression was measured by quantitative polymerase chain reaction (qPCR) and expressed as the fold change relative to control (CTL) (A and C), and proteins were detected by immunoblotting (B). D, Mouse embryonic fibroblasts were transfected with negative control small interfering RNA (siNeg) and small interfering RNA against β-catenin (siCat), TCF-4 (siTCF4), and LEF-1 (siLEF1) and then treated with or without 200 ng/ml recombinant mouse Wnt-3A for 48 hours. MMP-13 mRNA expression was measured by qPCR. Bars in A, C, and D show the mean ± SD results for 3 donors, each tested in triplicate (A and C) and triplicate cultures (D). NS = not significant. ∗ = P < 0.05; ∗∗ = P < 0.01.

Previous studies suggested that the transcription factors TCF-4 and LEF-1 acting together with β-catenin might act as transcriptional repressors of target gene expression (28–30). We therefore examined the roles of TCF-4 and LEF-1 in regulating MMP expression. TCF-4 and LEF-1 were effectively depleted in human chondrocytes by lentiviral shRNA–mediated knockdown (see Supplemental Figures 2E–G, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). The knockdown of both genes showed minimal effects on MMP expression (Figure 4C), suggesting that the repression of MMP expression in human chondrocytes is not mediated by the conventional complex of β-catenin and TCF/LEF, which raises the possibility that MMP repression is mediated by β-catenin independently of its DNA binding partners, for example by cross-talk with other pathways involved in MMP expression. In contrast, β-catenin, TCF-4, and LEF-1 were required for Wnt-3A–induced MMP expression in mouse embryonic fibroblasts, because knockdown of all 3 genes eliminated the procatabolic effect of Wnt-3A on MMP-13 expression (Figure 4D and Supplemental Figures 2H–K [available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/]), suggesting involvement of the β-catenin–TCF/LEF complex in the regulation of MMP expression in mouse cells (31).

Suppression of MMP expression through an inhibitory interaction of β-catenin and NF-κB.

It has been shown that IL-1β–induced expression of MMP-1 and MMP-13 in human chondrocytes is dependent on NF-κB activation (7–9). In addition, an interaction between β-catenin and NF-κB in various human cell types has recently been described (32–34). We therefore sought to determine whether an interaction between NF-κB and β-catenin might explain β-catenin–mediated inhibition of MMP expression in human chondrocytes. In agreement with previous studies (7–9), knockdown of NF-κB p65/RelA abrogated the expression of basal and IL-1β–induced MMP-1 and MMP-13 mRNA expression (Figure 5A); knockdown of NF-κB p50, the active form of NFKB1, diminished only expression of MMP-13, while MMP-1 mRNA expression was increased (Supplemental Figures 3A and B [available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). This increase in MMP-1 mRNA expression was not reflected in an increase in MMP-1 protein expression, which is most likely explained by a lag phase between an increase in mRNA expression and protein expression. MMP-3 expression was regulated by both p50 and p65 (see Supplemental Figure 3C, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/).

Figure 5.

Beta-catenin suppresses MMP expression through an inhibitory interaction with NF-κB. A, MMP mRNA was assessed by qPCR, and protein expression was detected by immunoblotting in human chondrocytes transduced with lentiviruses expressing scrambled shRNA and shRNA against NFKB1 (shNFKB1) and RelA (shRelA), after treatment with 10 ng/ml interleukin-1β (IL-1β) for 24 hours. Quantitative PCR data are expressed as the fold change relative to untreated scrambled shRNA–transduced cells as control (n = 1 donor tested in triplicate). B, Human chondrocytes were transduced with lentiviruses expressing an NF-κB reporter and Renilla luciferase alone or together with lentiviruses expressing scrambled shRNA and shRNA against β-catenin. Chondrocytes were treated with 100 ng/ml recombinant human Wnt-3A, 5 ng/ml IL-1β, or both for 24 hours, and luciferase reporter activity was measured. Data are expressed as the fold change relative to control (CTL) (n = 3 donors, each tested in triplicate). ∗∗ = P < 0.01. C, Protein lysates of human chondrocytes stimulated with 100 ng/ml recombinant human Wnt-3A, 5 ng/ml IL-1β, or both for 48 hours were isolated and subjected to immunoprecitation (IP) using NF-κB p65 antibody as the bait protein. Coimmunoprecipitated proteins were detected by immunoblotting. D, Human chondrocytes were treated with 200 ng/ml recombinant human Wnt-3A, 10 ng/ml IL-1β, or both for 48 hours. IL6 and SERPINA1 mRNA expression was measured by qPCR (n = 4 donors, each tested in triplicate). ∗ = P < 0.05; ∗∗ = P < 0.01. NS = not significant (see Figure 4 for other definitions).

We next examined the effect of Wnt-3A on NF-κB activity, using lentiviral transduction of a NF-κB–responsive promoter reporter construct in human chondrocytes. Treatment with Wnt-3A significantly decreased basal and IL-1β–induced NF-κB reporter activity (Figure 5B). This effect was dependent on β-catenin, because knockdown of β-catenin abolished a Wnt-3A–induced decrease in NF-κB promoter reporter activity and enhanced IL-1β–induced reporter activation (Figure 5B). Using a coimmunoprecipitation assay, we observed that β-catenin was able to form a protein complex with NF-κB p65 in human chondrocytes under basal conditions (Figure 5C). The interaction between β-catenin and NF-κB p65 was weakened upon activation of NF-κB by IL-1β and was strengthened after Wnt-3A–induced stabilization of β-catenin. Our data suggest that activation of NF-κB signaling by IL-1β in human chondrocytes may require dissociation of β-catenin from the NF-κB signaling complex. Coimmunoprecipitation of β-catenin with NF-κB p65 was also observed in bovine chondrocytes and mouse embryonic fibroblasts (see Supplemental Figure 4A, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). Wnt-3A was able to decrease basal and IL-1β–induced NF-κB activity in mouse embryonic fibroblasts (see Supplemental Figure 4B, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/), suggesting that the interaction between β-catenin and NF-κB alone may not explain the species-related differentiation in the regulation of MMP expression by Wnt/β-catenin signaling.

We next investigated whether canonical Wnt signaling could down-regulate other NF-κB target genes. We tested this hypothesis by evaluating the effect of Wnt-3A treatment on expression of IL6 and SERPINA1, 2 established target genes of the NF-κB pathway, in human chondrocytes (see Supplemental Figure 3C, available at http://www.utwente.nl/tnw/dbe/publications/2012/ma/). The mRNA expression of both genes was up-regulated by IL-1β but down-regulated by stimulation with Wnt-3A (Figure 5D). Wnt-3A treatment was also able to decrease IL-1β–induced IL-6 mRNA expression but failed to significantly counteract the effect of IL-1β on SERPINA1 mRNA expression (Figure 5D). These results suggest that Wnt/β-catenin signaling negatively regulates a subset of NF-κB target genes, most likely via an inhibitory protein–protein interaction with NF-κB.

DISCUSSION

Accumulating evidence, based mainly on experimental animal models of OA, has suggested that Wnt/β-catenin signaling has an important role in the pathogenesis of OA by driving, among other things, hypertrophic differentiation of chondrocytes and the expression of matrix-degrading MMPs in articular cartilage. Here, we propose that this hypothesis requires revisiting in human OA. In remarkable contrast to what has been observed in animal models, we provide evidence that Wnt/β-catenin signaling is a potent inhibitor of MMP-1, MMP-3, and MMP-13 mRNA and protein expression in human chondrocytes and is part of a negative feedback loop counteracting procatabolic NF-κB signaling activated by proinflammatory cytokines. Thus, in human articular cartilage, β-catenin may have an anticatabolic role by inhibiting MMP expression, as opposed to its established procatabolic role in animal cartilage. Similar observations were made in human chondrocytes from patients with OA, patients with RA, and healthy adult donors as well as in human fetal chondrocytes and adult bone marrow–derived MSCs, indicating that the inhibitory response of Wnt/β-catenin on MMP expression is conserved across various human mesenchymal cell types, irrespective of age and disease status.

It has been shown that IL-1β induces Wnt-7A expression and β-catenin accumulation in rabbit chondrocytes (13). We did not detect significant Wnt-7A expression in human chondrocytes. However, we did observe that IL-1β increased Wnt-7B expression and decreased expression of several Wnt inhibitors in human chondrocytes. Together, the concerted action of the increase in Wnt-7B expression and the decrease in Wnt inhibitor expression may be responsible for the indirect increase in β-catenin protein levels after IL-1β stimulation. Differential expression of Wnt proteins has previously been observed in joint diseases. In particular, Wnt-7B was shown to be significantly up-regulated in OA cartilage (17). We showed that overexpression of Wnt-7B repressed MMP expression, while knockdown of Wnt-7B enhanced MMP expression. This implies that up-regulation of Wnt-7B, as observed in OA cartilage, may be considered an attempt of human chondrocytes to reduce the expression of MMPs in order to slow down matrix degradation. Moreover, we provide evidence that Wnt-7B might be a driving factor in an anticatabolic negative feedback loop induced by procatabolic IL-1 signaling, although we cannot exclude roles for other canonical Wnts. In addition, IL-1β–induced loss of Wnt inhibitor expression is another important mechanism for negative feedback on MMP expression, because Dkk-1 enhanced both basal and IL-1β–induced MMP expression. These inhibitors may act independently of the change in Wnt-7B, or, based on time course experiments showing that the IL-1β–induced increase in Wnt-7B expression coincides with a decrease in the expression of Wnt antagonists, may potentiate the effect of increased Wnt-7B expression.

It has been shown previously that the interaction of β-catenin with TCF/LEF transcription factors can either stimulate or repress transcription in a promoter- and context-dependent manner (28–30). In mouse chondrocytes, β-catenin and TCF/LEF transcription factors are required for induction of MMP-13 expression (31). Indeed, knockdown of either TCF-4 or LEF-1 abrogated Wnt-3A–induced MMP expression in mouse embryonic fibroblasts. The observation that knockdown of TCF-4 or LEF-1 did not relieve the β-catenin–mediated repression of MMP expression in human chondrocytes clearly demonstrates that the canonical transcriptional function of β-catenin is not involved in inhibiting MMP expression in these cells. These data for mouse and human cells pinpoint the species-specific regulation of MMP expression to a differential role of TCF/LEF transcription factors in the regulation of MMP expression. The reason why humans have lost the propensity to regulate MMP-3 and MMP-13 expression by the β-catenin–TCF/LEF transcription complex remains elusive.

Previous studies have shown that β-catenin inhibits NF-κB–mediated signaling in human cancer cells via a protein–protein interaction between β-catenin and NF-κB (32–34). In human chondrocytes, it is well known that activation of NF-κB signaling by proinflammatory cytokines such as IL-1β potently induces cartilage matrix degradation by stimulating the expression and activity of various MMPs. We therefore sought to determine whether cross-talk between β-catenin and NF-κB could explain the inhibitory effects of canonical Wnt signaling on MMP expression in human chondrocytes. We extended the observations made in cancer cells to human chondrocytes and showed that activated β-catenin signaling can inhibit several NF-κB target genes. We furthermore showed that activation of NF-κB weakens the protein–protein interaction, while stabilization of β-catenin strengthens the interaction between NF-κB and β-catenin. These data strongly suggest that the TCF/LEF-independent repressive effect of β-catenin on MMP expression is mediated by negative cross-regulation of the NF-κB signaling pathway.

Remarkably, coimmunoprecipitation assays using mouse embryonic fibroblasts and bovine chondrocytes demonstrated the presence of a protein complex between NF-κB and β-catenin in these cells. In addition, we showed that Wnt-3A was able to decrease NF-κB reporter activity in mouse cells. These data suggest that mouse β-catenin was able to inhibit NF-κB–mediated signaling, and that this interaction cannot explain the species difference in regulation of MMP expression by canonical Wnt signaling. Our results suggest that in animal cells, the canonical Wnt/β-catenin signaling pathway via TCF/LEF prevails over the inhibitory effects on NF-κB signaling in the regulation of MMP-3 and MMP-13 expression; this contrasts remarkably with human cells, in which the noncanonical effect on NF-κB is dominant.

There has been longstanding debate regarding whether animal models are suited for studying the pathogenesis of OA, and it is believed that none of the current models can recapitulate all facets of human disease. Our observation of differential regulation of MMP expression by Wnt/β-catenin signaling may explain part of this discrepancy. Although the Wnt/β-catenin pathway is evolutionarily conserved, our data indicate that it is able to activate distinct subsets of target genes in a species-dependent manner that is at least partly explained by differential use of canonical and noncanonical β-catenin signaling. We propose that differential use of cross-talk of signaling networks may be responsible for species differences in cellular responses to Wnt and other extracellular signaling pathways (26, 35). Our findings suggest that animal models may not be suited for studying the role of Wnt/β-catenin signaling in human OA and raise the question of whether strategies aimed at inhibiting β-catenin in chondrocytes will be successful in the management of OA.

Recently, it was shown that Wnt-3A can modulate the human articular chondrocyte phenotype by activating both β-catenin–dependent and β-catenin–independent pathways (27). In line with the findings of our study, Nalesso et al (27) showed that chondrocyte proliferation and inhibition of MMP-3 expression are dependent on β-catenin. In contrast, effects of Wnt-3A on the expression of chondrocyte markers are independent of β-catenin. These data are supported by the observation that noncanonical Wnt-5A potently inhibits COL2A1 expression and induces MMP expression (36, 37). Taken together, the data suggest prominent roles for both canonical and noncanonical Wnt signaling in articular chondrocytes, although the canonical pathway might not be a key factor in the pathogenesis of human OA.

In summary, our study revealed an unexpected and novel role of Wnt/β-catenin signaling in human articular chondrocytes with respect to the expression of MMPs (Figure 6). During OA development, β-catenin signaling is up-regulated in human cartilage. This is at least partly attributable to increased Wnt-7B expression and/or down-regulation of Wnt inhibitors and can be further augmented by the activity of proinflammatory cytokines such as IL-1β. Accumulated β-catenin in turn represses NF-κB activity and consequently represses the expression of MMPs in human chondrocytes. Thus, Wnt/β-catenin signaling is part of a negative feedback loop counteracting procatabolic NF-κB in OA.

Figure 6.

A Wnt/β-catenin negative feedback loop inhibits interleukin-1β (IL-1β)–induced MMP expression in human chondrocytes. A, In animal cartilage, IL-1β indirectly activates canonical Wnt signaling by up-regulation of Wnt ligands. Subsequently, the transcription complex of β-catenin with TCF/LEF induces the expression of MMP-3 and MMP-13, leading to cartilage destruction. B, In human articular cartilage, IL-1β up-regulates MMP-1, MMP-3, and MMP-13 expression predominantly through activation of the NF-κB pathway, resulting in cartilage degradation. Simultaneously, IL-1β indirectly activates β-catenin through up-regulation of Wnt-7B expression and down-regulation of canonical Wnt inhibitors such as Dkk-1, Frizzled-related protein, and Wnt inhibitory factor 1. Stabilized β-catenin interacts with and inhibits NF-κB, which leads to the suppression of MMP expression in a negative feedback loop. It remains unclear whether the β-catenin pathway is directly involved in human cartilage degeneration. See Figure 4 for other definitions.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Karperien 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 conception and design. Ma, van Blitterswijk, Karperien.

Acquisition of data. Ma, Karperien.

Analysis and interpretation of data. Ma, Karperien.

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