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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

To determine the roles of the hedgehog and Wnt signaling pathways in accumulation of superficial zone protein (SZP) in surface zone articular chondrocytes.

Methods

Explant cultures of disks of surface zone cartilage or isolated chondrocytes from the surface zone of articular cartilage of bovine stifle joints were cultured in serum-free chemically defined medium. Accumulation of SZP in the culture medium, in response to hedgehog proteins (sonic hedgehog [SHH] and Indian hedgehog [IHH]), Wnt proteins (Wnt-3a, Wnt-5a, and Wnt-11), agonists of the Wnt/β-catenin pathway (glycogen synthase kinase 3β [GSK-3β] inhibitors), and antagonists of the Wnt/β-catenin pathway, was investigated. The interaction between transforming growth factor β1 (TGFβ1) and hedgehog proteins or antagonists of the Wnt/β-catenin pathway was also investigated.

Results

Hedgehog proteins stimulated SZP accumulation. Activation of the Wnt/β-catenin pathway by Wnt-3a and GSK-3β inhibitors led to inhibition of SZP accumulation; however, Wnt-5a and Wnt-11 had no influence on SZP accumulation. Conversely, antagonists of the Wnt/β-catenin pathway stimulated SZP accumulation. In addition, there were combinatorial effects of TGFβ1 and hedgehog proteins or antagonists of the Wnt/β-catenin pathway on SZP accumulation.

Conclusion

SHH and IHH signaling has a stimulatory effect on SZP accumulation in surface zone cartilage and isolated articular chondrocytes. These findings provide insight into the regulatory mechanisms of articular cartilage homeostasis and maintenance by morphogens.

Lubrication of articular cartilage is critical for normal joint function. Superficial zone protein (SZP), homologous to lubricin and proteoglycan 4 (PRG4) (1, 2), is a large proteoglycan that is synthesized and secreted into synovial fluid by chondrocytes in the surface zone of articular cartilage and by synovial cells (3–6). SZP is known to function as a boundary lubricant in articular cartilage and reduces the coefficient of friction (7–9).

In addition to its function as a boundary lubricant, SZP has been shown to have other biologic functions, such as cell proliferation, cytoprotection, and matrix binding (2, 10). Both SZP and lubricin are encoded by the Prg4 gene (9), and mutations in the Prg4 gene can result in camptodactyly-arthropathy–coxa vara–pericarditis syndrome, an autosomal recessive disease that leads to alteration of the articular surface and attendant degradation of articular cartilage, causing early-onset noninflammatory joint damage and failure (11, 12). Furthermore, SZP inhibits synovial cell overgrowth and protects articulating surfaces from protein and cell adhesion and infiltration (13).

Loss of SZP influences the functional properties of the synovial joints, and a focal decrease in SZP in early osteoarthritis (OA) could have a role in the pathogenesis of cartilage degeneration (13, 14). Elsaid et al demonstrated, in an experimental rabbit model of arthritis, that there was a strong association between loss of the boundary-lubricating abilities of synovial fluid and damage to the articular cartilage after joint injury (15). Taken together, these findings suggest that SZP plays an essential role in maintaining healthy joint function and homeostasis.

It is well established that cytokines play important roles in cartilage homeostasis and that SZP is regulated, in part, by the cytokines involved in cartilage homeostasis in the joints (1, 10). Previous studies demonstrated that the level of SZP secreted into the medium can be regulated by different cytokines (5, 6, 10, 16), including transforming growth factor β (TGFβ), a critical regulator of SZP accumulation in surface zone articular chondrocytes.

There is growing recognition of several novel signaling pathways in articular cartilage (17–19). Hedgehog and Wnt signaling play key roles in skeletal development, including effects on chondrogenesis via the regulation of cell proliferation, differentiation, survival, and migration (17, 20–22). A novel role for morphogens of the hedgehog and Wnt families in synovial joint formation has been proposed (21, 23–25). In addition, the hedgehog and Wnt signaling pathways have been implicated in the pathogenesis of OA (19, 26). However, the actions of hedgehog and Wnt signaling on surface zone cartilage, and the articular chondrocytes therein, have not been investigated. We therefore hypothesized that hedgehog and Wnt signaling might regulate SZP accumulation in the surface zone of the articular cartilage.

The hedgehog gene was first identified in Drosophila melanogaster and has a role in embryonic segment polarity (27). In mammals, there are 3 hedgehog orthologs, sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (22). SHH and IHH have distinct and overlapping roles in embryonic development (22), and IHH plays a central role in coordinating growth and differentiation of chondrocytes through the formation of a negative feedback loop with parathyroid hormone (PTH)–related protein (PTHrP) in the developing endochondral skeleton (28, 29). In the absence of hedgehog ligands, Patched-1 (Ptch-1) represses the activity of Smoothened (Smo), which mediates all vertebrate signaling (30). Binding of hedgehog ligands to the Ptch-1 receptor releases the inhibitory effects of Ptch-1 on Smo, and thereby allows Smo signaling to process the glioma associated oncogene homolog (Gli) family of transcription factors, which up-regulate downstream target genes (22).

The Wnt proteins are a family of highly conserved secreted glycoproteins that mediate receptor-mediated signaling pathways. There are 19 mammalian homologs of Wnts (25, 31). The name Wnt is derived from a combination of Drosophila wingless and mouse Int (32). Wingless was originally identified as a segment polarity gene in D. melanogaster and was shown to be homologous to Int1, a gene identified as an oncogene. Wnts are implicated in embryogenesis and adult limb formation during mouse development (33). Signal transduction of Wnts is well scrutinized, and the most well-understood pathway is the Wnt/β-catenin pathway, also known as the canonical pathway. In this pathway, in the absence of Wnt ligands, β-catenin, the main mediator of the signal relay, is bound in a destruction complex composed of glycogen synthase kinase 3β (GSK-3β), Axin, adenomatous polyposis coli gene product, and other interacting proteins. As a result, β-catenin is phosphorylated and degraded, resulting in low cytosolic β-catenin levels (18).

Some members of the Wnt family, such as Wnt-1 and Wnt-3a, bind to Frizzled receptors and the coreceptors low-density lipoprotein receptor–related protein 5 (LRP-5) and LRP-6, and inhibit GSK-3β–mediated phosphorylation of β-catenin (31, 34). Stabilized β-catenin then accumulates in the cytosol and translocates to the nucleus, where it interacts with lymphoid enhancer factor/T cell–specific transcriptional factor to affect transcription (18, 35). In contrast, other Wnts, such as Wnt-5a and Wnt-11, can, instead, activate β-catenin–independent Wnt pathways, generally referred to as noncanonical pathways, such as the planar cell polarity pathway and Wnt/Ca2+ pathways (36).

The aim of this study was to investigate the roles of hedgehog and Wnt signaling in SZP accumulation in surface zone articular chondrocytes, using primary cell cultures and disks of cartilage explants. Specifically, we investigated the influence of 2 hedgehog proteins (SHH and IHH), PTHrP, PTH(1–34), and 3 different Wnt proteins (Wnt-3a, Wnt-5a, and Wnt-11) on SZP accumulation. In addition, the influences of agonists and antagonists of the Wnt/β-catenin pathway on SZP accumulation were determined. We also examined the interactions between TGFβ1, which is a critical regulator of SZP accumulation in surface zone articular chondrocytes, and hedgehog or Wnt signaling.

MATERIALS AND METHODS

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

Cell isolation and culture.

Surface zone bovine articular chondrocytes were obtained as described previously (6). Briefly, stifle (knee) joints from 3-month-old calves were obtained within 6 hours of slaughter from a local abattoir. The joints were dissected under aseptic conditions, exposing the femoral condyles. The surface zone of the articular cartilage was harvested from the anterior half of the lateral and medial femoral condyles (∼100 μm thick) using a dermatome. The cartilage slices were divided into small pieces with a razor blade, and digested with 0.2% collagenase P (Roche) in Dulbecco's modified Eagle's medium (DMEM) along with a nutrient mixture of DMEM/F-12 (Gibco) containing 50 μg/ml ascorbate-2-phosphate (Sigma-Aldrich), 0.1% bovine serum albumin (BSA; Sigma-Aldrich), and antibiotics (Medium-A) with 3% fetal bovine serum (FBS) (Gibco) for 2 hours at 37°C. The released chondrocytes from the tissues were filtered through a cell strainer (70 μm; BD Falcon) and rinsed with DMEM/F-12.

Isolated chondrocytes were plated in monolayers, at a density of 1 × 105 cells/well (∼2.5 × 104 cells/cm2), in 12-well culture plates (Corning) in Medium-A with 10% FBS, and incubated at 37°C in a moist atmosphere of 5% carbon dioxide and 95% air. After 24-hour equilibration in the culture medium, cells were switched to fresh Medium-A with ITS+ Premix (insulin–transferrin–selenium; BD Biosciences) containing various concentrations of hedgehog proteins, Wnt proteins, other proteins, or chemical compounds.

Human recombinant proteins SHH, IHH, Wnt-11, sclerostin (SOST), dickkopf-1 (Dkk-1), and TGFβ1 were purchased from R&D Systems. PTHrP, PTH(1–34), and GSK-3β inhibitors (lithium chloride [LiCl], BIO, SB216763, and SB415286) were purchased from Sigma-Aldrich. Mouse Wnt-3a and Wnt-5a were generous gifts from Dr. R. Nusse (Stanford University, Stanford, CA). Although hedgehog and Wnt proteins are highly conserved within and across species (22, 31), we also performed GenBank protein BLAST analysis for sequence comparisons between human and bovine amino acid identities, in order to investigate their potential cross-reactivity. Human SHH (N-terminal domain), IHH (N-terminal domain), Wnt-11, SOST, Dkk-1, and TGFβ1 share 93%, 100%, 98%, 93%, 91%, and 100% amino acid identity, respectively, with the bovine proteins. Mouse Wnt-3a and Wnt-5a share 97% and 99% amino acid identity, respectively, with the bovine proteins (results not shown). The protein concentrations chosen were based on previously determined doses (37–40).

Cyclopamine (Sigma-Aldrich), an inhibitor of hedgehog signaling that acts by directly binding to Smo (41), was used as an antagonist of hedgehog signaling. Cells were pretreated with cyclopamine (1 μM or 10 μM) for 3 hours before the addition of SHH (1 μg/ml).

Tissue acquisition for explant culture.

Previously, we reported that SZP expression varies across different surface regions of the lateral and medial femoral condyles (9). Therefore, in order to minimize possible variations in SZP surface distribution, cartilage explants were obtained from only the central region of the lateral femoral condyle of each joint, as described previously (5). Two osteochondral plugs were obtained in close proximity, using a 5-mm–diameter punch (Acuderm). The surface 300-μm layer of each plug was then separated using a custom-built slicing device. From these 5-mm–diameter cartilage disks, 2-mm–diameter disks were obtained using a 2-mm–diameter punch (Acuderm). Cartilage disks (2 mm diameter) were plated at 1 disk per 1 ml medium per well in 12-well culture plates in Medium-A with 10% FBS, and incubated at 37°C in a moist atmosphere of 5% carbon dioxide and 95% air. After 24-hour equilibration in the culture medium, cells were switched to fresh Medium-A with ITS+ Premix (BD Biosciences) containing SHH (1 μg/ml), Wnt-3a (100 ng/ml), or SOST (3 μg/ml).

Enzyme-linked immunosorbent assay (ELISA) for SZP.

Since it is known that the majority of SZP is secreted into the culture medium (3), the medium from the cultures was harvested after the 4-day treatment and quantitatively analyzed for SZP by sandwich ELISA, using purified SZP as a standard (6). Each well of 96-well MaxiSorp plates (Nalge Nunc International) was coated with 1 μg/ml peanut lectin (EY Laboratories) in 50 mM sodium carbonate buffer (pH 9.5). The wells were then blocked with 1% BSA in the same buffer. Aliquots of culture medium were incubated in the wells.

Thereafter, the wells were incubated with monoclonal antibody S6.79 (1:5,000; a generous gift from Dr. T. Schmid, Rush Medical College, Chicago, IL) as the primary antibody and goat anti-mouse IgG conjugated with horseradish peroxidase (1:3,000; Bio-Rad) as the secondary antibody. SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was added, and the results were quantified in a luminometer (expressed as relative luminescence units). Between all steps, the wells were washed with phosphate buffered saline containing 0.05% Tween 20 (Sigma). SZP levels were calculated using an SZP standard, which was purified by affinity chromatography on a peanut lectin column (3, 42); purity was verified by immunoblot analysis and quantified using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific) (5, 6, 43).

Protein extraction and immunoblot analysis.

After treatment with Wnt-3a (100 μg/ml) or BIO (1 μM) for 6 hours, the cultured cells were washed twice with phosphate buffered saline and lysed in buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS). The cell lysates were sonicated and then centrifuged at 14,000 revolutions per minute for 10 minutes. Protein concentrations were determined with a Micro BCA Protein Assay Kit.

For immunoblot analysis, equal quantities of protein extracts were resolved by SDS–polyaclylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk in TBST (25 mM Tris HCl, 125 mM NaCl, and 0.1% Tween 20) for 1 hour and incubated overnight at 4°C with rabbit monoclonal antibodies against β-catenin or β-actin (as loading control) (Cell Signaling Technology) at a 1:1,000 dilution. The membrane was then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:3,000 dilution; Bio-Rad) for 1 hour, followed by a 1-minute incubation with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), which was developed for visualization.

Real-time polymerase chain reaction (PCR) analysis.

After the 4-day treatment, total RNA was extracted from the monolayer cells using an RNeasy Mini kit (Qiagen) or from the cartilage explants using a Polytron tissue homogenizer (Brinkman Instruments) and an RNeasy Lipid Tissue Mini kit (Qiagen), with on-membrane DNase I (Qiagen) digestion to avoid genomic DNA contamination. Total RNA was reverse transcribed into single-strand complementary DNA (cDNA) using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time PCR was performed in triplicate on the cDNA using an Applied Biosystems 7900HT Fast Real-Time PCR System and SYBR Green reagents, in accordance with the manufacturer's recommended protocols. Results were normalized to GAPDH levels and expressed relative to the control (untreated) culture levels (using the ΔΔCt method; Applied Biosystems). The forward and reverse primer pairs used in this study were as follows: for GAPDH, 5′-GGCGCCAAGAGGGTCAT-3′ and 5′-GTGGTTCACGCCCATCACA-3′; for Ptch1, 5′-TGCCCAGGCTACGAGGACTA-3′ and 5′-CCGGACATTAAAAGGCACATG-3′; for Gli1, 5′-TGCACATGCGCAGACACACG-3′ and 5′-ACTGCAGCCCTCATGCTCACAC-3′; for hedgehog-interacting protein gene (HHIP), 5′-TAAGCTGCTGCTGTTGGCCG-3′ and 5′-TTTTTCAGGCGCTTCGGGGG-3′; and for AXIN2, 5′-TCCTGCTGCGCCAAGTGCAA-3′ and 5′-TGGCGCTGTGCTTCGTCTTG-3′.

Immunohistochemical analysis of cartilage tissue for SZP.

Cartilage disks after the 4-day treatment were fixed in Bouin's solution (Sigma) for 6 hours, followed by paraffin embedding and sectioning at 5 μm. Immunostaining was performed according to standard methods, using monoclonal antibody S6.79 (1:5,000 dilution) as the primary antibody (9, 42) and an avidin–biotin–peroxidase kit (Vector Laboratories) with mouse IgG secondary antibody for signal detection.

Statistical analysis.

For all experiments, except for assessment of SZP accumulation in the explant cultures, the sample size was 6, representing 6 different animals. For explant cultures, the sample size was 16, representing 16 different animals. Values are presented as the mean ± SD. For multiple comparisons (except for the analysis of interactive effects), a one-way analysis of variance (ANOVA) was performed, using the StatView statistical program (SAS Institute). If a significant difference was found, Tukey's post hoc test was conducted.

For analysis of interactive effects between TGFβ1 and SHH, IHH, SOST, or Dkk-1, we used two-way ANOVA. A paired 2-tailed t-test was performed to determine the difference between the control group and treatment group in explant cultures. 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

Influence of hedgehog proteins on SZP accumulation.

Hedgehog proteins were added to the primary cell cultures of bovine articular chondrocytes at concentrations of 0.1, 0.3, and 1 μg/ml. Both SHH and IHH significantly stimulated the accumulation of SZP in monolayer cultures, at all 3 protein concentrations. Compared to the untreated control, treatment of the articular chondrocytes with SHH at a concentration of 1 μg/ml elicited the maximum level of increase in SZP, a 2.4-fold increase, while 1 μg/ml IHH produced a 1.7-fold increase (Figure 1A). In contrast, PTHrP and PTH(1–34) had no influence on SZP accumulation (results not shown).

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Figure 1. Influence of hedgehog proteins on superficial zone protein (SZP) accumulation. A and B, Surface zone articular chondrocytes were cultured in monolayer for 4 days in serum-free chemically defined medium with graded levels (0.1, 0.3, and 1 μg/ml) of sonic hedgehog (SHH) or Indian hedgehog (IHH) (A), while surface zone cartilage explants were cultured for 4 days with 1 μg/ml SHH (B). SZP accumulation in the culture medium was quantified by enzyme-linked immunosorbent assay. Bars show the mean ± SD SZP levels in 6 samples (A) or 16 samples (B) per group. C, SZP accumulation was also assessed by immunohistochemical staining of the cartilage explants, following treatment with 1 μg/ml SHH. Untreated samples served as control. ∗ = P < 0.01; ∗∗ = P < 0.05 versus untreated control. Bars = 100 μm.

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We also investigated the effects of 1 μg/ml SHH on the surface layer of bovine articular cartilage in explant cultures. Similar to the findings in monolayer cultures, SHH stimulated the accumulation of SZP in medium from explant cultures, with a significant difference compared to the untreated control (Figure 1B). In addition, the explants treated with 1 μg/ml SHH for 4 days exhibited increased SZP signal intensity at the surface layer (Figure 1C).

Dependence of SZP accumulation on Smo in hedgehog signaling.

We first examined the expression of the hedgehog target genes Ptch1, Gli1, and HHIP (19). Both SHH and IHH up-regulated the expression of all 3 hedgehog target genes after treatment of the cells with either 0.3 or 1 μg/ml of SHH or IHH (for HHIP, all 3 concentrations of SHH had an effect) in monolayer cultures (Figure 2A).

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Figure 2. Hedgehog target gene expression in monolayer cells and cartilage extracts, and dependence of SZP accumulation on Smoothened (Smo) in hedgehog signaling. A and C, After 4 days of stimulation with various concentrations of SHH or IHH in the culture medium of monolayer cells (A) or cartilage explants (C), total RNA was extracted and reverse transcribed into cDNA, with results assessed by real-time polymerase chain reaction. Values are the mean ± SD fold increase in expression of the hedgehog target genes Ptch1, Gli1, and HHIP in treated samples relative to untreated controls (n = 6 samples per group). B, Dependence of SZP accumulation on Smo in hedgehog signaling was assessed by pretreating the primary cells for 3 hours with cyclopamine (Cyc) (1 or 10 μM), an inhibitor of hedgehog signaling via direct binding to Smo, before the addition of 1 μg/ml SHH (compared to cultures without SHH). Cells were then cultured in monolayer for 4 days, and SZP accumulation in the medium was quantified by enzyme-linked immunosorbent assay. DMSO was used as a vehicle control. Values are the mean ± SD levels of SZP in 6 samples per group. = P < 0.01; ∗∗ = P < 0.05 versus untreated or vehicle-treated control. See Figure 1 for other definitions.

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We then investigated the mechanism underlying the action of hedgehog proteins on SZP accumulation. As Smo is a critical signal transducer of the hedgehog signaling pathway, we used an inhibitor of Smo, cyclopamine, to determine the mechanism. The up-regulation of SZP accumulation by 1 μg/ml SHH in primary cell cultures was reduced, in part, by pretreatment of the cells with 1 μM cyclopamine, and was completely abolished by cyclopamine at a dose of 10 μM (Figure 2B). However, treatment with cyclopamine alone showed no influence on SZP accumulation. Similar to the findings in monolayer cultures, SHH up-regulated the expression of the hedgehog target genes in cartilage explant cultures (Figure 2C).

Influence of Wnt proteins on SZP accumulation.

Bovine articular chondrocytes were treated with 3 different Wnt proteins at concentrations of 10, 30, and 100 ng/ml. Wnt-3a, which can activate the Wnt/β-catenin pathway, suppressed the accumulation of SZP in a dose-dependent manner, with significant differences compared to the untreated control. In contrast, Wnt-5a and Wnt-11 had no influence on SZP accumulation (Figure 3A). Similar to the findings in monolayer cultures, 100 ng/ml Wnt-3a significantly suppressed SZP accumulation in the medium from explant culture (Figure 3B). However, immunostaining for SZP in the cartilage explants treated with 100 ng/ml Wnt-3a for 4 days did not show discernible changes when compared to the untreated control explants (results not shown).

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Figure 3. Influence of Wnt proteins and glycogen synthase kinase 3β (GSK-3β) inhibitors on SZP accumulation. Surface zone articular chondrocytes were cultured for 4 days in serum-free chemically defined medium with graded levels (10, 30, and 100 ng/ml) of Wnt proteins (Wnt-3a, Wnt-5a, and Wnt-11) (A) or with the indicated concentrations of GSK-3β inhibitors (lithium chloride [LiCl], BIO, SB415286, and SB216763) (C), and surface zone cartilage explants were cultured for 4 days with 100 ng/ml Wnt-3a (B). SZP accumulation in the medium was quantified by enzyme-linked immunosorbent assay. Untreated or DMSO-treated samples were used as controls. Results are the mean ± SD SZP levels in 6 samples (A and C) or 16 samples (B) per group. ∗ = P < 0.01; ∗∗ = P < 0.05 versus untreated or DMSO-treated control. See Figure 1 for other definitions.

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Inhibitory effect of the Wnt/β-catenin pathway on SZP accumulation.

We next examined the inhibitory effect of the Wnt/β-catenin pathway on SZP accumulation. For this experiment, we utilized the GSK-3β inhibitors LiCl, BIO, SB415286, and SB216763, whose inhibitory effects are known to lead to the accumulation of β-catenin and the activation of the Wnt/β-catenin pathway. All 4 of the GSK-3β inhibitors significantly suppressed the accumulation of SZP in cultures of surface zone articular chondrocytes, in comparison with the SZP levels in untreated and vehicle-treated control cultures (Figure 3C).

Activation of the Wnt/β-catenin pathway in the cells following treatment with either Wnt-3a or the GSK-3β inhibitor BIO was confirmed by our observations of the accumulation of the cytosolic protein β-catenin (Figure 4A). In addition, Wnt-3a up-regulated the expression of AXIN2, a target gene of the Wnt/β-catenin pathway, in both monolayer and explant cultures (Figures 4B and C). These results indicated that activation of the Wnt/β-catenin pathway can lead to suppression of SZP accumulation.

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Figure 4. Accumulation of β-catenin in monolayer cultures of bovine articular chondrocytes after treatment with Wnt-3a or BIO, and expression of AXIN2, a target gene of the Wnt/β-catenin pathway, in both monolayer cultures and explant cultures. A, After treatment with or without Wnt-3a (100 ng/ml) or BIO (1 μM) for 6 hours, whole cell lysates were collected and analyzed by immunoblotting for β-catenin. Results shown are representative of 3 individual experiments. β-actin was used as loading control. B and C, Expression of the Wnt/β-catenin pathway target gene AXIN2 was assessed in monolayer cultures of primary cells (B) or in cartilage extracts (C) after 4 days of stimulation with the indicated concentrations of Wnt-3a. Total RNA was extracted from the monolayer cells or cartilage explants and reverse transcribed into cDNA, with results analyzed by real-time polymerase chain reaction. Values are the mean ± SD fold expression in treated samples relative to untreated controls (n = 6 samples per group). = P < 0.01; ∗∗= P < 0.05 versus untreated control.

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Increased SZP accumulation after inhibition of the Wnt/β-catenin pathway.

Since we found that activation of the Wnt/β-catenin pathway suppressed the accumulation of SZP, we performed experiments with SOST and Dkk-1, both of which are antagonists of the LRP-5/LRP-6 coreceptors and inhibit the Wnt/β-catenin pathway. Both SOST (at concentrations of 1 and 3 μg/ml) and Dkk-1 (at concentrations of 0.1, 0.3, and 1 μg/ml) significantly stimulated the accumulation of SZP in monolayer cultures. Compared with the untreated control, the maximum increase in SZP levels was 2-fold when the cells were treated with 3 μg/ml SOST, and the maximum increase was 1.5-fold when the cells were treated with 0.3 μg/ml Dkk-1 (Figure 5A).

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Figure 5. Increase in SZP accumulation following inhibition of the Wnt/β-catenin pathway. A and B, Surface zone articular chondrocytes were cultured in monolayer for 4 days in serum-free chemically defined medium with antagonists of the low-density lipoprotein receptor–related protein 5 (LRP-5)/LRP-6 coreceptors (sclerostin [SOST] or dickkopf-1 [Dkk-1]) in various concentrations (A), and surface zone cartilage explants were cultured for 4 days with 3 μg/ml SOST (B). SZP accumulation in the medium was quantified by enzyme-linked immunosorbent assay. Bars show the mean ± SD SZP levels in 6 samples (A) or 16 samples (B) per group. C, SZP accumulation was also assessed by immunohistochemical staining of the cartilage explants, following treatment with 3 μg/ml SOST. Untreated samples served as control. = P < 0.01; ∗∗= P < 0.05 versus untreated control. Bars = 100 μm. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Similar to the findings in monolayer cultures, 3 μg/ml SOST stimulated the accumulation of SZP in the medium from cartilage explant cultures, with significant differences compared to the untreated control (Figure 5B). In addition, the explant treated with 3 μg/ml SOST for 4 days exhibited moderately increased SZP signal intensity at the surface layer (Figure 5C). However, treatment with SOST at a concentration of 1 μg/ml did not result in any discernible changes in SZP accumulation or SZP staining in explant cultures (results not shown).

Cell proliferation assay.

To determine the influence of SHH, Wnt-3a, and SOST on cell proliferation, we performed a cell proliferation assay in which we conducted cell counts on a hemocytometer with trypan blue exclusion. There were no significant differences in cell number after 4-day culture between the untreated control cultures and the cultures treated with 1 μg/ml SHH, 100 ng/ml Wnt-3a, or 1 μg/ml SOST (Figure 6A).

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Figure 6. Influence of SHH, Wnt-3a, and sclerostin (SOST) on proliferation of bovine articular chondrocytes, and synergistic effects of transforming growth factor β1 (TGFβ1) and hedgehog proteins or antagonists of the Wnt/β-catenin pathway on SZP accumulation in monolayer or explant cultures. A, Cell proliferation assays were performed in 4-day monolayer cultures by counting the number of cells on a hemocytometer with trypan blue exclusion. Bars show the mean ± SD in 6 samples per group (SHH-, Wnt-3a–, or SOST-treated cultures compared with untreated control cultures). B and C, Synergistic effects of TGFβ1 (10 ng/ml) and hedgehog proteins (SHH or IHH) or antagonists of the Wnt/β-catenin pathway (SOST or dickkopf-1 [Dkk-1]) were assessed in monolayer cultures of primary cells (B) or in explant cultures (C). Surface zone articular chondrocytes were cultured with or without TGFβ1 and treated with or without SHH, IHH, SOST, or Dkk-1 (A), while surface zone cartilage explants were cultured with or without TGFβ1 and treated with or without SHH or SOST (B). SZP accumulation in the medium after the 4-day treatment was quantified by enzyme-linked immunosorbent assay. Values are the mean ± SD in 6 samples (B) or 16 samples (C) per group. See Figure 1 for other definitions.

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Synergistic effects of TGFβ1 and hedgehog proteins or antagonists of the Wnt/β-catenin pathway.

Finally, we examined the interaction between TGFβ1 (10 ng/ml) and SHH (1 μg/ml), IHH (1 μg/ml), SOST (1 μg/ml), or Dkk-1 (0.3 μg/ml) in monolayer cultures, and between TGFβ1 (10 ng/ml) and SHH (1 μg/ml) or SOST (3 μg/ml) in explant cultures. In monolayer cultures, TGFβ1 and SHH, TGFβ1 and IHH, TGFβ1 and SOST, and TGFβ1 and Dkk-1 synergistically increased the accumulation of SZP in the medium (P = 0.0002, P = 0.02, P = 0.0002, and P = 0.01, respectively, for each interaction term) (Figure 6B). However, in explant cultures, synergistic effects were not observed for either the TGFβ1–SHH interaction (P = 0.82) or the TGFβ1–SOST interaction (P = 0.52). Rather, the results suggested that these interactions had additive effects on the accumulation of SZP in cartilage explants (Figure 6C).

DISCUSSION

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

The results of the present investigation demonstrate the actions of the hedgehog or Wnt signaling pathways on surface zone articular chondrocytes. Hedgehog proteins stimulated SZP accumulation. Activation of the Wnt/β-catenin pathway by Wnt-3a and GSK-3β inhibitors led to the inhibition of SZP accumulation. It is noteworthy that Wnt-5a and Wnt-11 were devoid of any influence on SZP accumulation. Conversely, inhibitors of the Wnt/β-catenin pathway stimulated SZP accumulation.

The hedgehog signaling pathway is involved in the initiation of synovial joint formation, endochondral ossification, articular cartilage differentiation and maintenance, and the pathogenesis of OA (19, 21, 23, 44). IHH plays a role in skeletal development, particularly in the growth plate (28), and is closely related to SHH, which is the main regulator of limb outgrowth (45). Previously, in a study by Lin et al, it was demonstrated that hedgehog signaling is activated in OA, and higher levels of hedgehog signaling in articular chondrocytes cause a more severe OA phenotype (19). However, little is known about the detailed action of hedgehog proteins on normal articular chondrocytes. In the present study, hedgehog proteins (SHH and IHH) stimulated SZP accumulation in both monolayer and explant cultures. This finding indicates that the surface zone of articular cartilage may be regulated by hedgehog signaling. Since IHH and PTHrP form a negative feedback loop in the growth plate, in which IHH stimulates the production of PTHrP by periarticular chondrocytes, we also investigated the influence of PTHrP and PTH(1–34) on SZP accumulation. However, neither PTHrP nor PTH(1–34) was found to have an influence on the surface zone chondrocytes.

Wnt signaling pathways also play key roles in synovial joint formation and have been implicated in not only cartilage homeostasis, but also the pathogenesis of OA (24–26). It has been observed that several proteins have either catabolic or anabolic effects on chondrocytes. Wnt-3a has a catabolic effect on articular chondrocytes and activates the Wnt/β-catenin pathway (38), while Wnt-5a has a catabolic effect and activates a β-catenin–independent pathway (37, 46). In contrast, Wnt-11 has an anabolic effect on articular cartilage and activates a β-catenin–independent pathway (46). In the present study, Wnt-3a and GSK-3β inhibitors suppressed the accumulation of SZP in the cell culture medium by activating the Wnt/β-catenin pathway, which is consistent with the findings in previous studies (38). In explant cultures, Wnt-3a did not decrease the signal intensity of SZP at the surface of the articular cartilage (results not shown), but this might be because the majority of the synthesized SZP was secreted into the culture medium (5), and also because the signal intensity of SZP in the control culture was low. Meanwhile, in the present study, both Wnt-5a and Wnt-11 had no influence on the accumulation of SZP, which is in contrast to findings in previous studies (37, 46).

Mechanical loading is also a critical factor in articular cartilage homeostasis, especially with regard to its effects on the surface zone. It is noteworthy that mechanical shear stimulates SZP accumulation in the surface zone of articular cartilage (9, 47). Recently, a novel role of Wnt/β-catenin signaling as a mediator of the effects of mechanical loading on cartilage homeostasis was observed. It was demonstrated that activation of Wnt/β-catenin signaling by Wnt-3a repressed the mechanical loading–induced up-regulation of chondrocyte phenotype markers such as aggrecan and SOX9 (48). In the present investigation, Wnt-3a repressed the accumulation of SZP in surface zone articular chondrocytes. Therefore, it is possible that inhibition of SZP accumulation by Wnt/β-catenin signaling may have some relevance to mechanical loading. Further investigation is required to validate this hypothesis.

We also demonstrated that inhibition of the Wnt/β-catenin pathway by SOST and Dkk-1 (antagonists of the LRP-5/LRP-6 coreceptors) led to the stimulation of SZP accumulation in monolayer and explant cultures. In explant cultures, treatment with SOST at a dose of 3 μg/ml stimulated the accumulation of SZP (Figures 5B and C), but this was not evident at a dose of 1 μg/ml (results not shown). A previous study showed that articular chondrocytes express low levels of β-catenin, a key mediator of the Wnt/β-catenin signaling pathway, and β-catenin levels are significantly increased during de-differentiation of articular chondrocytes in serial monolayer cultures (49). Therefore, inhibition of this low level of endogenous Wnt/β-catenin signaling in surface zone articular chondrocytes may lead to the stimulation of SZP accumulation. This may also be one possible reason for the different findings between monolayer and explant cultures.

There were synergistic effects between TGFβ1 and hedgehog proteins and between TGFβ1 and antagonists of the Wnt/β-catenin signaling pathway in primary cell monolayer cultures. In contrast, in explant cultures, the effects of TGFβ1 and SHH and of TGFβ1 and SOST were additive rather than synergistic. This may be due to the difference in culture conditions. Even so, these findings still reveal the importance of these signals in the regulation of SZP accumulation. Previous studies have demonstrated an interaction between TGFβ1 and Wnt signaling or between hedgehog and Wnt signaling in skeletal development, including endochondral bone and synovial joint formation (21, 50). Therefore, further investigations of the interactions among these signals in terms of their association with the accumulation of SZP in surface zone articular chondrocytes will be needed.

Relatively little is known regarding the role of hedgehog and Wnt/β-catenin signaling in the maintenance of adult articular cartilage in vivo. Several studies have suggested that both hedgehog signaling and Wnt signaling are more activated in OA cartilage than in healthy articular cartilage (19, 26). The hedgehog antagonist cyclopamine, when added alone, had no effect on the basal level of SZP accumulation, suggesting that hedgehog signaling is not critical in surface zone articular cartilage.

This study has some limitations. Although we found that activation of hedgehog signaling led to the stimulation of SZP accumulation in bovine articular chondrocytes, it has been reported that, in mouse models of OA, higher levels of hedgehog signaling in articular chondrocytes may cause a more severe OA phenotype (19). However, those investigators utilized transgenic mice, a model in which the activation of hedgehog signaling or the doses of hedgehog proteins were higher than those in the present study. In addition, as articular cartilage is a heterogeneous tissue, consisting of surface, middle, and deep zones, further experiments are needed to assess the effect of hedgehog signaling on each zone in the presence of various protein concentrations.

In conclusion, the present investigation provides novel insights into the role of the hedgehog and Wnt signaling pathways in accumulation of SZP in the surface zone of articular cartilage. The information in the present study may be helpful in developing new tissue-engineering strategies to repair and regenerate the surface zone of articular cartilage, with the goal of achieving optimal lubrication of the joints and restoring normal joint function.

AUTHOR CONTRIBUTIONS

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

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. Reddi 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. Iwakura, Reddi.

Acquisition of data. Iwakura, Inui.

Analysis and interpretation of data. Iwakura, Reddi.

Acknowledgements

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

We thank Dr. R. Nusse (Stanford University, Stanford, CA) for his generous gifts of the Wnt-3a and Wnt-5a proteins, and Dr. T. Schmid (Department of Biochemistry, Rush Medical College, Chicago, IL) for his generous gift of the monoclonal antibody S6.79.

REFERENCES

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