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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Osteoarthritis Cartilage. 2017 Jun 3;25(9):1505–1515. doi: 10.1016/j.joca.2017.05.018

Wnt5a Induces Catabolic Signaling and Matrix Metalloproteinase Production in Human Articular Chondrocytes

G Huang †,§, S Chubinskaya ‡, W Liao §, RF Loeser †,*
PMCID: PMC5565712  NIHMSID: NIHMS886069  PMID: 28587781

Abstract

Objective

Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA.

Design

Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting.

Results

Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the JNK and ERK pathways. Wnt5a reduced aggrecan gene expression after 48 hours of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated β-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production.

Conclusions

Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.

Keywords: Wnt, chondrocyte, metalloproteinase, integrin, cell signaling

Introduction

The progressive destruction of articular cartilage during the development of osteoarthritis (OA) is thought to be a consequence of excess chondrocyte catabolic activities including increased production of matrix metalloproteinases (MMPs)1–3. A variety of soluble mediators, including various chemokines, cytokines, fibronectin fragments (FN-f) and Wnt family members produced locally by articular chondrocytes and cells in neighboring joint tissues, act in an autocrine and paracrine fashion to increase additional proinflammatory factors and MMPs4. FN-f have been found at relatively high levels in synovial fluid and cartilage from OA patients5. These matrix fragments stimulate chondrocyte production of MMPs, inflammatory cytokines and chemokines through multiple signaling pathways, including mitogen-activated protein kinases (MAPKs), resulting in cartilage degradation both in vitro and in vivo suggesting that FN-f could play an important role in the development of OA6–8. However, the underlying mechanisms responsible for regulating MMP production by other mediators, such as Wnt proteins, are not completely understood.

The Wnt family can be divided into two categories: the canonical class which activates the β-catenin dependent pathway and the non-canonical class which activates β-catenin independent pathways9. In the canonical Wnt pathway, binding of a Wnt ligand to the Frizzled (FZD) receptor inhibits glycogen synthase kinase 3 β (GSK-3β)-mediated β-catenin phosphorylation, leading to stabilization and nuclear translocation of β-catenin to activate transcription of Wnt target genes. In contrast, the non-canonical Wnt pathway is activated through FZD receptors or other non-class FZD receptors such as receptor tyrosine kinase-like orphan receptor 1 (ROR1), ROR2, and receptor-like tyrosine kinase, and functions independent of β-catenin9. Two of the best characterized non-canonical Wnt signaling pathways are the Wnt/Ca2+ and Wnt/planar cell polarity (PCP) pathways which activate calmodulin-dependent kinase II (CaMKII) and c-Jun N-terminal kinase (JNK), respectively. Wnt signaling plays an important role in various developmental processes including cartilage and bone formation and increasing evidence suggests that aberrant Wnt signaling contributes to cartilage destruction in OA10, 11.

Wnt5a is a representative ligand of the non-canonical Wnt class and is one of the most extensively studied Wnt proteins9. Wnt5a has been shown to regulate cartilage development by promoting chondrocyte differentiation and inhibiting chondrocyte maturation12. Moreover, Wnt5a expression was detected at increased levels in osteoarthritic tissues including cartilage, inflamed synovial membrane and acetabular labrum13–15. We previously discovered Wnt5a as the most upregulated Wnt family member in a network analysis with time series microarray data from joints of mice with OA induced by destabilization of the medial meniscus (DMM)16. However, a role for Wnt5a in human chondrocytes or in cartilage degeneration during development of OA has not been reported. The purpose of the current study was to investigate the potential role of Wnt5a in OA using human articular chondrocytes and determine the signaling pathways involved.

Methods

Antibodies and Reagents

Antibodies to Wnt5a/b, phospho-β-catenin(Ser33/37/Thr41), phospho-CaMKII(Thr286), phospho-JNK(Thr183/Tyr185), phospho-p38(Thr180/Tyr182), phospho-ERK(Thr202/Tyr204), phospho-p65, phospho-Akt(Ser473), phospho-GSK-3β(Ser9), phospho-Smad1(Ser463/465)/ Smad5(Ser463/465)/ Smad8(Ser465/467), phospho-Smad1(Ser206), phospho-Smad2(Ser465/467), and total β-catenin, CaMKII, JNK, p38, ERK, p65, Akt, GSK-3β and Smad1, and secondary antibodies were from Cell Signaling Technology. Antibodies to MMP2, MMP3 and MMP13 were from EMD Millipore. MMP1 antibody was from Abnova. β-actin antibody was from Abcam. Wnt5a antibody (Cat. No. AF645) for immunohistochemistry (IHC), recombinant Wnt5a protein and recombinant Wnt3a protein were from R&D Systems. Purified endotoxin-free recombinant 42kDa FN-f, containing the cell binding RGD domain, was produced as previously described17. RT2 qPCR primers were from Qiagen except that aggrecan (ACAN) primer was as previously described18. ImProm-II™ Reverse Transcriptase and SsoAdvanced™ Universal SYBR® Green Supermix were from Promega and Bio-Rad, respectively. Chemical inhibitors were from Calbiochem (JNK-IN-8, SB203580, LY294002, Box-5, CaMK IINtide), Cell Signaling Technology (PD98059, U0126), and Selleck Chemicals (Ruxolitinib).

Primary Chondrocyte Isolation and Culture

Normal human cartilage was obtained from tali of tissue donors through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL) and the Department of Pediatrics at Rush University Medical Center (Chicago, IL). The donors had no known history of joint disease. The tissue was graded as described19 and only the normal cartilage with Collin’s grade 0–1 or normal appearing cartilage from joints with Collins’s grade 2 was used. The chondrocytes were obtained from a total of 27 individual donors. The ages of tissue donors ranged from 22 to 70 years.

Chondrocytes were isolated by sequential enzymatic digestion as previously described20. Unless specifically indicated, cells were cultured in DMEM/F12 media supplemented with 10% fetal bovine serum and antibiotics in high density monolayers until confluent. All cells were used without passaging to ensure maintenance of chondrocyte phenotype and were changed to serum-free conditions prior to use in experiments.

Chondrocyte Stimulation

Cells were stimulated with 1 µM FN-f and/or 200 ng/ml Wnt5a protein for the indicated time with or without pretreatment with signaling inhibitors. Inhibitor concentrations were as follows: JNK1/2 (JNK-IN-8, 10 µM), p38 (SB203580, 10 µM), MEK1/2 (PD98059, 30 µM or U0126, 10 µM), JAK1/2 (ruxolitinib, 10 µM), PI-3 Kinase (LY294002, 5 µM), CaMK II (CaMK IINtide, 1.25–5 µM) and Wnt-5a (Box-5, 50–400 µM). The inhibitors were added 30 minutes before stimulation except JNK-IN-8 and Box-5 which were added 4 hours and 40 minutes prior to stimulation, respectively. Treatment with 1 µM FN-f for 30 minutes and 100 ng/ml recombinant Wnt3a protein for 60 minutes were used as positive controls for activation of MAPK signaling and canonical Wnt signaling, respectively.

Immunohistochemistry

Human cartilage sections were a kind gift from Dr. Martin Lotz (Scripps Research Institute, La Jolla, CA). The sections were from young (ages 36–48 years, n=4), aged (ages 68–76 years, n=4) and OA (ages 64–90 years, n=4) donors. The sections were deparaffinized in xylene and rehydrated in serial ethanol washes followed by antigen retrieval in sodium citrate at 95 °C for 20 minutes. After blocking with 3% H2O2 (Fisher Scientific) and Protein Block (DAKO) at room temperature, the sections were incubated overnight at 4 °C with goat anti-Wnt5a antibody (1:15 dilution in DAKO antibody diluent). Tissue incubated with antibody diluent without primary antibody was used as a negative control. The following day, Goat-on-Rodent HRP-Polymer (Biocare Medical) was applied to recognize the primary antibody and Liquid DAB + Substrate Chromagen System (DAKO) was used for stain development. The sections were counterstained with Harris Hematoxylin (Sigma).

Quantitative real-time PCR (RT-qPCR)

Total RNA was isolated from primary human chondrocytes using Total RNA Purification Plus Kit (Norgen Biotek, Canada) following the manufacturer’s instructions. RNA concentration and quality was determined by NanoDrop 2000 (Thermo Scientific). Total RNA was converted to cDNA by ImProm-II™ Reverse Transcriptase (Promega) with oligo (dT) and random hexamer primers. The cDNA was diluted to a final concentration of 5ng/µl and analyzed by RT-qPCR utilized SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) on Bio-Rad CFX96 Real-Time System. A total of 20 µl RT-qPCR reaction volume included 10 µl of SYBR Green supermix (2×), 0.8 µl of RT2 qPCR Primer (10 µM), 7.2 µl of Nuclease-free H2O and 2 µl of template cDNA. The reactions were performed on a CFX96 Real-time PCR System (Bio-Rad, USA) as follows: 50 °C for 2 minutes, then 95 °C for 10 minutes, followed by 41 cycles of 95 °C for 15 seconds, and 60 °C for 1 minute. Melt curve analysis was performed from 60 to 95 °C with increments of 0.5 °C. Amplification was conducted in duplicate for each sample. Gene expression was normalized to housekeeping gene TBP and calculated using the 2−ΔΔCq method.

Immunoblotting

Cells were washed and lysed as previously described21. The soluble protein concentration was determined using Pierce Micro BCA Kit (Thermo Scientific). Samples with equal amount of total protein were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the Wnt5a primary antibody or phosphospecific antibodies followed by stripping and re-probing with antibodies to β-actin or the corresponding total protein, as a loading control, as described21.

For conditioned media, equal volumes of media were run on SDS-PAGE and used for immunoblotting for MMPs and Wnt5a as above. Specially, for analysis of Wnt5a secretion level, conditioned media were concentrated (10:1) by the 10K Amicon® Ultra Centrifugal Filters (EMD Millipore). The levels of MMP2, which were found not to change with FN-f treatment in our previous studies22, were used as a loading control in experiments of FN-f treatment. Band density was quantified using ImageJ software. All immunoblotting experiments were repeated at least three times with chondrocytes from different donors.

Statistical Analysis

The data were analyzed using GraphPad Prism version 7 (GraphPad Software, Inc.). The results are presented as mean values ± 95% confidence interval (CI) from a minimum of three independent biological replicates. For the qPCR data, a log2 transformation was performed to have a decrease shown in fold below zero. The exact number of independent samples used for each experiment is provided in the figure legends. Significant differences were evaluated by Wilcoxon matched-pairs signed rank test for comparison between two groups or Friedman test followed with Dunn’s multiple comparison test for multi-group comparisons. A p-value < 0.05 was considered significant.

Results

Wnt5a is present in articular cartilage with OA changes

Sections of human tibial cartilage from young adults, older adults and older OA patients were subjected to immunohistochemical staining with Wnt5a antibody. Low levels of chondrocyte intracellular staining were detected in normal appearing cartilage sections from young and older adults (Fig. 1, A–D). Matrix and cellular staining for Wnt5a was observed in lesion areas from older adult cartilage (Fig. 1, E and F) that appeared to be stronger in OA cartilage (Fig. 1, G and H).

Fig. 1.

Fig. 1

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Immunohistochemical staining for Wnt5a in young normal (A, B), aged normal (C, D), aged lesion (E, F) and osteoarthritic (G, H) human tibial cartilage. Representative images from four different donors for each group are shown. The panels on the right are the magnified images of the regions in boxes in the corresponding panels on the left. Scale bar, 200 µm in panel A, C, E, G and 50 µm in panel B, D, F, H.

Wnt5a expression and secretion are upregulated by FN-f stimulation in human chondrocytes

Since Wnt5a was present in cartilage with OA-like damage, we examined whether human chondrocytes could produce Wnt5a in response to a catabolic stimulus. We used normal human chondrocytes treated with recombinant FN-f as a model of catabolic stimulation. After 24 hours stimulation, FN-f significantly increased Wnt5a RNA levels but not Wnt5b, Wnt4 or Wnt11 RNA in human chondrocytes (Fig. 2A). Wnt5a protein levels in cell lysates (Fig. 2C) and conditioned media (Fig. 2D) were also increased by FN-f as determined by immunoblotting with an antibody for Wnt5a/b. Since Wnt5b RNA did not change in response to FN-f, we considered the band detected on the immunoblots to be Wnt5a although the antibody recognizes both Wnt5a and Wnt5b.

Fig. 2.

Fig. 2

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Wnt5a RNA expression and protein production is induced by FN-f. A. Normal human chondrocytes were treated with 1 µM FN-f for 24 hours. Total RNA was isolated from chondrocytes of six different donors (34–64 years old) and mRNA expression of Wnt5a, Wnt5b, Wnt4 and Wnt11 were measured by RT-qPCR. B–D. Normal human chondrocytes from three different donors (B: 46–64 years old, C: 48–53 years old, D: 22–53 years old) were pre-treated with 10 µM JNK-IN-8 for 4 hours or other inhibitors (10 µM SB203580, 10 µM ruxolitinib, 30 µM PD98059 and 5 µM LY294002) for 30 minutes followed by 24 hours stimulation with 1 µM FN-f. Wnt5a mRNA levels were determined by RT-qPCR. Wnt5a protein levels in total cell lysates (C) and concentrated (1:10) conditioned media (D) were determined by immunoblotting with Wnt5a/b antibody. Levels of β-actin and MMP2 were used as loading controls. For RNA data, expression of Wnt5a and Wnt5b mRNA were normalized to TBP mRNA expression and calculated by 2−ΔΔCq method and shown as relative change compared with untreated control. Data with log2 transformation are presented as individual data points with the mean indicated by the horizontal line and 95% CI indicated by the error bars. For panel A, the mRNA expression of Wnt in the control group was set as 1 and displayed as 0 with a log2 transformation. Wilcoxon matched-pairs signed rank test was conducted to evaluate the differences between FN-f treatment and untreated controls (panel A) and Friedman test with Dunn’s multiple comparison test was used for comparison in multiple groups (panel B). A p-value < 0.05 was considered significant. The exact p values are shown on the graph. For protein expression, representative blots of three independent experiments are shown.

In order to determine the mechanism underlying Wnt5a upregulation by FN-f, we used different pathway inhibitors to investigate effects of signaling inhibition on FN-f induced Wnt5a expression and protein production. The highly specific JNK inhibitor, JNK-IN-8, significantly blocked FN-f stimulated Wnt5a mRNA expression while the MEK1/2 inhibitor PD98059 and the general PI-3 Kinase inhibitor LY294002 partially inhibited Wnt5a mRNA expression that was not statistically significant (Fig. 2B). Consistent with RNA data, inhibitors for JNK and MEK1/2 significantly suppressed FN-f induced Wnt5a protein production and secretion while inhibitors for p38 and PI-3 Kinase only showed a slight suppression on Wnt5a protein production and secretion (Fig. 2, C and D). We did not see any effects of the JAK1/2 inhibitor ruxolitinib on Wnt5a expression or protein production stimulated by FN-f.

Wnt5a inhibits anabolic gene expression and promotes MMP production in human chondrocytes

As Wnt5a was found to be present in OA cartilage and in normal chondrocytes stimulated with FN-f, we tested purified recombinant Wnt5a protein for effects on anabolic and catabolic gene expression. RNA levels of ACAN were reduced after 48 hours of treatment with Wnt5a while COL2A1 was also decreased but did not reach statistical significance due to variability among the donor samples tested (Fig. 3, A and B). Meanwhile, RNA levels of MMP1, 3 and 13 were enhanced with the time of Wnt5a incubation but the differences were only significant for MMP-1 at the 24 hour time point compared with untreated controls (Fig. 3, C–E). Results from immunoblotting showed secretion of MMP protein was also increased by Wnt5a in a time-dependent manner (Fig. 3F). MMP1 and MMP13 production increased gradually in response to Wnt5a with significant levels detected at 24 or 48 hours while MMP3 was present in media from unstimulated cells and did not significantly increase with addition of Wnt5a. MMP2, which was not changed with FN-f treatment (Fig. 4A), was induced by Wnt5a within 1 hour of treatment.

Fig. 3.

Fig. 3

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Effects of Wnt5a on expression of anabolic and catabolic genes in normal human chondrocytes. Chondrocytes in monolayer were treated with 200 ng/ml recombinant Wnt5a protein for 1, 3, 6, 24 and 48 hours. RNA and conditioned media were collected. A–E, Expression of ACAN, COL2A1, MMP1, MMP3, and MMP13 mRNA were normalized to TBP mRNA expression and calculated by 2−ΔΔCq method and shown as relative change compared with untreated controls. Data with log2 transformation are presented as individual data points with the mean value indicated by the horizontal line and 95% CI indicated by the error bars from four independent donors (48–69 years old). The exact p values shown on the graph represent significant differences compared with time 0 evaluated by Friedman test with Dunn’s multiple comparison test. F. MMP1, MMP13, MMP3 and MMP2 protein in conditioned media were measured by immunoblotting. Results are representative of experiments performed with cells from four different donors (45–65 years old).

Fig. 4.

Fig. 4

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Role of Wnt5a in FN-f induced MMP production in normal human chondrocytes. A. Human monolayer chondrocytes were pretreated with increasing doses of Box-5, a specific inhibitor for Wnt5a, for 40 minutes followed by 24 hours stimulation with 1 µM FN-f. Conditioned media were collected and immunoblotted for MMP1, MMP13 and MMP3. MMP2 was used as a loading control. Representative blots of three independent experiments using chondrocytes from three individual donors (45–64 years old) are shown. B, C. Densitometric analysis of immunoblots from three independent donors. Relative band intensity was calculated by normalizing to loading control and FN-f treatment. Data are presented as individual data points with the mean value indicated by the horizontal line and 95% CI by the error bars. D. Chondrocytes were stimulated for 24 hours with 1 µM FN-f, 100 ng/ml Wnt5a or a combination of FN-f and Wnt5a. Conditioned media were collected for immunoblotting with MMP1, MMP13 and MMP3 antibodies. MMP2 was used as a loading control. Representative immunoblots from three independent experiments using chondrocytes from three individual donors (43–60 years old) are shown.

Wnt5a does not appear to be required for FN-f induced MMP13 production

We next determined the role of Wnt5a in FN-f induced MMP production. Normal human chondrocytes were pre-treated with increasing doses of Box-5, a specific peptide inhibitor for Wnt5a, followed by 24 hours of FN-f stimulation. MMP1 and MMP13 protein in conditioned media were significantly increased by FN-f, while MMP3 and MMP2 production were not changed. The Wnt5a inhibitor did not reduce MMP1 levels in response to FN-f (Fig. 4B) while the highest dose (400 µM) showed a 35.9% decrease in FN-f induced MMP13 production (Fig. 4C).

The findings imply that Wnt5a is not likely required for FN-f induced MMP1 or MMP13 production but perhaps Wnt5a could promote further MMP13 production in response to FN-f. We tested this by co-stimulation using a dose of Wnt5a (100 ng/ml) that does not increase MMP production by itself. Co-stimulation with Wnt5a did not promote a further increase above FN-f alone (Fig. 4D).

Cell signaling responses to Wnt5a in human chondrocytes

In order to understand the downstream signaling in response to Wnt5a in human chondrocytes, we examined effects of Wnt5a on canonical and non-canonical Wnt signaling pathways evaluated by immunoblotting in a time course analysis. Representative blots are shown in Figure 5 and densitometric analysis from at least three independent donors are shown in Figure 6. Canonical Wnt signaling is associated with reduced levels of β-catenin phosphorylation. We noted that Wnt3a, used as a positive control, but not Wnt5a, reduced the levels of phosphorylated β-catenin (Fig. 5A). Wnt5a caused a modest increase in phosphorylation of β-catenin at 90 minutes, which suggested that Wnt5a may inhibit canonical Wnt signaling through increased β-catenin phosphorylation (Fig. 5A and Fig. 6A). Wnt5a stimulated weak phosphorylation of CaMKII with a peak of 1.48-fold at 15 and 30 minutes (Fig. 5A and Fig. 6B). Although it was much weaker than FN-f used as a positive control, treatment with Wnt5a resulted in mildly elevated levels of phosphorylated JNK and statistical analysis revealed that the increase was significant at 90 minutes (Fig. 5A and Fig. 6C).

Fig. 5.

Fig. 5

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Time course of chondrocyte signaling in response to Wnt5a. Confluent human chondrocytes cultured in serum-free media were stimulated with 200 ng/ml Wnt5a for the indicated times (0–90 minutes). Chondrocytes treated with 1 µM FN-f for 30 minutes or 100 ng/ml Wnt3a for 60 minutes were used as positive controls for activation of MAPK signaling and canonical Wnt signaling, respectively. Total cell lysates were immunoblotted with antibodies against phosphorylated signaling proteins. Blots were then stripped and reprobed with respective total antibodies or β-actin for loading controls. All immunoblots are representative of at least three independent experiments performed using cells from different tissue donors (36–69 years old, n=3–6). Strong bands for phosphorylated protein (p-JNK, p-p65 and p-Akt) in the FN-f treated cells or Wnt3a treated cells reduced the ability of the total antibody to recognize the total protein. A. Effect of Wnt5a on canonical and non-canonical Wnt signaling. The antibody to phosphorylated JNK appears to cross react with phosphorylated ERK, when there is strong phosphorylation of ERK relative to JNK, so in order to interpret the phosphorylation status of JNK accurately, only the upper band (54 kDa) in p-JNK blots was evaluated. B. Effect of Wnt5a on MAPK signaling and NF-κB signaling. C. Effect of Wnt5a on PI3K/Akt signaling. D. Effect of Wnt5a on Smad signaling. Due to the inconsistent results of total Smad1 in different donors, β-actin was used as a loading control for phosphorylated Smad proteins.

Fig. 6.

Fig. 6

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Densitometric analysis of time course of chondrocyte signaling in response to Wnt5a. Data from the experiments shown in Fig. 5 plus independent biological replicates was used. The band density of the phosphorylated protein was normalized to the total protein as a loading control (A–H), while the band density of the phosphorylated Smad protein was normalized to β-actin due to variability in total Smad1 in different donors (I–K). The results are presented as relative fold change from time 0 untreated controls. Data are shown as individual data points with the mean value indicated by the horizontal line and 95% CI indicated by the error bars from at least three independent experiments using different tissue donors. The exact p values shown on the graph represent significant differences compared with time 0 evaluated by Friedman test with Dunn’s multiple comparison test.

Compared to FN-f, Wnt5a treatment led to weak phosphorylation of p38, ERK, and p65 that peaked early (10–15 minutes) for p38 and ERK and later (60–90 minutes) for p65 (Fig. 5B and 6, D–F). For comparison, Wnt3a also stimulated p38 and ERK but not p65.

Akt and GSK-3β are the key kinases in PI3K/Akt signaling and their phosphorylation correlates with activation of this signaling pathway. Wnt5a stimulated relatively strong Akt phosphorylation starting at 5 minutes which on average peaked at around 15 minutes (Fig. 5C and 6G). Likewise, phosphorylation of GSK-3β was increased and this was significant at 5, 10 and 15 minutes (Fig. 5C and 6H). Wnt3a also stimulated phosphorylation of both Akt and GSK-3β.

We also investigated the effects of Wnt5a on Smad signaling, an important anabolic signaling pathway in chondrocytes. Wnt5a increased phosphorylation of the linker region (Ser206) of Smad1 after 15 minutes of treatment (Fig. 5D and 6J) which inhibits Smad1 activity. The effects of Wnt5a on phosphorylation of Smad1/5/8 and Smad2 were more variable and not statistically significant (Fig 5D and 6I and K). Addition of Wnt3a to cells did not alter phosphorylation of the Smads tested (Fig. 5D).

Inhibition of MAP kinases, PI-3 kinase and CaMKII suppress Wnt5a stimulated MMP production

To further determine the effects of Wnt5a signaling on MMP production, we pre-treated chondrocytes with inhibitors relevant to selected signaling pathways and measured MMP release in conditioned media by immunoblotting. Specific inhibition of the JNK, p38 and ERK MAP kinases and PI-3 kinase all reduced the amount of MMP1 and MMP13 produced in response to Wnt5a with the greatest reduction noted after inhibition of JNK (Fig. 7A). Two different inhibitors, U0126 and PD98059, were employed for inhibition of MEK1/2 to block ERK activation. U0126 showed a greater suppression on Wnt5a mediated MMP production than PD98059. Given the fact that U0126 is able to inhibit both MEK1 and MEK2 while PD98059 inhibits MEK1 more potently than MEK2, our data suggested that both MEK1 and MEK2 contributed to Wnt5a induced MMP1 and MMP13 production. The levels of MMP3 and MMP2 did not change in response to Wnt5a but the JNK inhibitor appeared to slightly reduce the level of MMP3.

Fig. 7.

Fig. 7

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Effects of signaling pathway inhibitors on Wnt5a induced MMP production. A. Confluent human articular chondrocytes were pre-treated with 10 µM JNK-IN-8, 10 µM SB203580, 10 µM U0126, 30 µM PD98059 or 5 µM LY294002 and then stimulated with 200 ng/ml recombinant Wnt5a protein for 24 hours. Conditioned media were collected and immunoblotted for MMP1, 13 and 3. MMP2 was used as a loading control. Representative blots of three independent experiments performed with three different tissue donors (24–53 years old) are shown. B. Confluent human articular chondrocytes from one 22-year-old donor were pre-treated with increasing doses of CaMK IINtide (1.25–5 µM) for 30 minutes followed by 24 hours stimulation of 200 ng/ml Wnt5a protein. Conditioned media were collected for immunoblotted with antibodies against MMPs. C. Confluent human chondrocytes were pre-treated with 5 µM CaMK IINtide for 30 minutes and then stimulated with 200 ng/ml Wnt5a protein for 24 hours. Conditioned media were collected for immunoblotted with antibodies against MMP1, 13, 3 and 2. Representative blots of three independent donors (47–69 years old) are shown. D. A schematic diagram of the proposed signaling pathways and their role in chondrocyte metabolic activity. All the solid lines indicate a pathway investigated in the current study or in previous studies in chondrocytes, while the dashed lines indicate steps in the pathways which require further study. Scale bar, 50 µm.

For the inhibition of CaMKII, we first performed a dose response experiment in human normal chondrocytes from one donor to determine the appropriate dose of the CaMKII inhibitor CaMKIINtide. The result showed that the higher concentration of CaMKIINtide (5 µM) obtained greatest inhibition on Wnt5a mediated MMP1 and MMP13 production (Fig. 7B). We then repeated the experiments in chondrocytes from another three donors using the 5 µM dose and confirmed inhibition of MMP1 and MMP13 but in some donors also noted reduced MMP3 and MMP2 (Fig. 7C).

Discussion

These results provide evidence that Wnt5a is present in human OA cartilage where it could promote chondrocyte catabolic signaling resulting in upregulation of MMP1 and MMP13 as well as inhibition of aggrecan expression. Chondrocyte expression and release of Wnt5a was stimulated by FN-f, a matrix fragment found in OA cartilage and synovial fluid that has been shown to stimulate catabolic signaling (reviewed in23, 24). Wnt5a was found to activate β-catenin independent Wnt signaling pathways, including activation of CaMKII, JNK, p38, ERK1/2, p65 and Akt. Production of Wnt5a in response to FN-f, as well as the production of MMP1 and MMP13 in response to Wnt5a, was regulated by multiple signaling pathways including MAPK and PI3K, suggesting their potential role in the actions of Wnt5a in cartilage (Figure 7D).

We previously found that RNA expression of Wnt5a was increased in the knee joint tissue of DMM mice as OA developed16. Similar to our findings, a recent study also reported the presence of Wnt5a protein in articular cartilage of knee OA patients by IHC13. An increase in Wnt5a expression induced by interleukin-1β (IL-1 β) in rabbit or rat chondrocytes has been reported which in rabbit chondrocytes required activity of NF-κB25, 26. We found that a second catabolic stimulus relevant to OA, FN-f, promoted Wnt5a expression and secretion in human articular chondrocytes and this effect could be blocked by specific inhibitors for JNK and MEK1/2 and partially suppressed by inhibitors for p38 and PI-3 kinase. The involvement of MAPK, JAK/STAT3 and PI3K/Akt signaling in regulation of Wnt5a have been reported in other cell types with different stimuli27–31. Our data, along with these previous reports, suggest that the regulation of Wnt5a is cell- and stimulus- specific.

FN-f treatment has been shown to induce expression of pro-inflammatory cytokines in chondrocytes that include IL-1β, IL-6 and TNF-α7, 32 that can induce Wnt5a expression26, 28, 33, suggesting that FN-f induction of Wnt5a could be mediated by one or more of these factors. However, we observed that Wnt5a expression increased as early as 6 hours after FN-f treatment (Fig. S1) which is similar to the time when other pro-inflammatory mediators begin to increase. Although, we cannot exclude the possibility that expression of Wnt5a by FN-f treatment results from activation of pro-inflammatory cytokine signaling, the timing suggests a direct response to FN-f.

A previous study showed that type II collagen expression was decreased in rabbit chondrocytes treated with Wnt5a conditioned medium25. We also oberved a reduction in type II collagen expression in human cells but due to variability among the donor samples it was not statistically significant while aggrecan expression was significantly reduced at the 48 hr time point. The anti-anabolic effect of Wnt5a could be due to the observed increased phosphorylation of the linker region of Smad1 (an inhibitory site) which would result in reduced BMP and/or TGF-β signaling.

Wnt5a has been shown to either activate or inhibit Wnt/β-catenin signaling depending on the receptor- and cell-context9, 34. We found that Wnt5a increased levels of phosphorylated GSK-3β at Serine 9 and β-catenin in human chondrocytes and did not stimulate β-catenin nuclear translocation (data not shown), indicating that canonical Wnt/β catenin signaling was not activated. This is consistent with the finding that Wnt-mediated inactivation of GSK3β is not dependent on phosphorylation of GSK-3β on Serine 942. However, whether there is a negative effect of Wnt5a on canonical signaling in human chondrocytes needs more direct evidence.

The finding of Wnt5a-induced phosphorylation of CaMKII and JNK were consistent with previous reports in rat and rabbit chondrocytes, respectively25, 35, 36. We found inhibitors for MAP kinases, PI-3 kinase and CaMKII significantly suppressed Wnt5a-stimulated MMP1 and MMP13 production consistent with non-canonical signaling. The crosstalk between Wnt signaling and MAPK, NF-κB and PI3K/Akt signaling has been proposed previously in other cell types37–41. In those studies, the patterns of protein phosphorylation in response to Wnt5a were similar to our data in that the phosphorylation of these signaling molecules was transient and weak.

Our study carries some limitations. The Box5 inhibitor used to block Wnt5a is a modified hexapeptide designed to be a ligand competitor rather than an inhibitor of a Wnt specific receptor43. However, we cannot exclude the possibility that Wnt5a signaling was not completely blocked by this inhibitor. The inhibitors used to block specific signaling proteins can have off-target effects. We used inhibitors extensively studied, either in our lab in previous studies or by other investigators, and shown to be the most specific available6, 18, 21, 36, 43–47. There are also limitations specific to human cell-culture studies. The variability among primary cells isolated from different human donors and the limited number of cells available for extensive experiments can reduce the chances of detecting significant results when differences are small but the advantage of using human cells over cell lines or cells from other species counters this limitation. Finally, demonstrating the presence of Wnt5a in OA cartilage is not sufficient to determine if it is active and capable of stimulating signaling.

In summary, Wnt5a is present in human articular cartilage with degenerative changes and with FN-f stimulation of normal chondrocytes. Wnt5a inhibits expression of aggrecan and promotes MMP production via multiple non-canonical signaling pathways. These results suggest that Wnt5a should be added to the list of Wnt family members that could play a role in promoting OA. Because of the perinatal lethality of homozygous Wnt5a knock-out in mice, further studies of the potential role of Wnt5a in vivo using conditioned deletion of Wnt5a in the joint are warranted.

Supplementary Material

supplement

Fig. S1. Expression of Wnt5a is stimulated with 6 hours treatment of FN-f. Normal human chondrocytes were treated with 1 µM FN-f for 6 hours (n=6). Wnt5a mRNA levels were determined by RT-qPCR and data with log2 transformation of relative fold change compared with untreated control are presented as mean ± 95%CI. Wilcoxon matched-pairs signed rank test was conducted for comparison between two groups. The exact p value was shown on the graph.

Acknowledgments

We thank Kathryn Kelley, Mary Zhao, Veronica Ulici and John Collins for technical assistance and Becki Cleveland for assistance with statistical analysis. We also thanks the Gift of Hope Organ and Tissue Donor Network (Itasca, IL) and the donor families for providing normal donor tissue and Dr. Martin Lotz for providing cartilage sections for immunohistochemistry.

Role of the funding sources

This study was funded by NIH grant R37AR049003 from the National Institute of Arthritis, Musculoskeletal, and Skin Diseases. G.H. was supported by a scholarship from the China Scholarship Council (No. 201506380076). Human tissue procurement was also supported by Rush Ciba-Geigy Endowed Chair (SC). The study sponsors did not have a role in the study design, collection, analysis or interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.

Footnotes

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Authors’ contributions
  1. Study conception and design: G.H. and R.F.L. Acquisition of Data: G.H. and S.C. Data analysis and interpretation: G.H., S.C., W.L. and R.F.L.
  2. Drafting of manuscript: G.H. and R.F.L. Revision of manuscript: all authors revised the manuscript critically for important intellectual content.
  3. Final approval of the manuscript: all authors have reviewed the final version of the manuscript and approved the version to be submitted.

Competing interests

There are no competing interests to disclose for any of the authors.

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Associated Data

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Supplementary Materials

supplement

Fig. S1. Expression of Wnt5a is stimulated with 6 hours treatment of FN-f. Normal human chondrocytes were treated with 1 µM FN-f for 6 hours (n=6). Wnt5a mRNA levels were determined by RT-qPCR and data with log2 transformation of relative fold change compared with untreated control are presented as mean ± 95%CI. Wilcoxon matched-pairs signed rank test was conducted for comparison between two groups. The exact p value was shown on the graph.

NIHMS886069-supplement.tif (340.5KB, tif)

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