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.
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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.