Explanations concerning the cause of osteoarthritis (OA) have long focused on the destruction of articular cartilage, the activating factors of which were thought to be triggered as a result of repetitive loading (1). Pathologic changes of cartilage in OA are associated with changes in the cellular phenotype of articular cartilage chondrocytes (ACCs) to a state of terminal differentiation (2, 3). However, the long-term molecular events that are responsible for this transition are not well understood.
Recent studies suggest that the subchondral bone plays a major role in OA cartilage changes, an indication of active communication between the subchondral bone and the cartilage in the progression of OA (4, 5). Bone anabolic factors, such as osteocalcin, osteopontin, and alkaline phosphatase (ALP) are all up-regulated in OA subchondral bone osteoblasts (SBOs) as compared with normal SBOs, supporting the notion of a dysfunction of osteoblast behavior (6–8). It has been shown in animal models of OA that a thickening of subchondral bone precedes cartilage changes (9, 10), and it has further been demonstrated that in vivo factors produced by OA SBOs increase glycosaminoglycan release from the cartilage (11) and can influence cartilage-specific gene expression (12). It was demonstrated by the application of a coculture model of bovine explant subchondral bone and cartilage that excision of subchondral bone from articular cartilage resulted in increased chondrocyte death, thus demonstrating the important role of subchondral bone in maintaining joint homeostasis (13). However, the molecular mechanisms, and, in particular, the signaling pathways, by which normal and OA SBOs regulate the articular cartilage phenotype remain unknown.
Activation of the 3 major classes of MAPKs (ERK-1/2, JNK, and p38 MAPK) has been detected in chondrocytes (14). MAPKs are known to be responsible for the conversion of a vast number of extracellular stimuli into specific cellular responses, including chondrocyte proliferation and differentiation (15, 16). The requirement of MAPK signaling pathways, in particular, p38 and ERK-1/2, during various phases of endochondral ossification has also been demonstrated in several studies (17, 18). MAPK signaling pathways have been shown to play a distinct role in aspects of cartilage biology, such as cartilage matrix synthesis and homeostasis (19, 20). The role of MAPK signaling in skeletal development and in the biology of cartilage points toward a possible association of altered MAPK signaling and OA. Indeed, alterations in these signaling pathways are reported to play a prominent role in chondrocyte dysfunction as a part of OA pathogenesis and disease progression (21).
Since the OA SBOs are reported to alter the cartilage phenotype, it is possible that these alterations in ACCs may occur via MAPK regulation. However, no studies to date have explored the role of MAPK signaling factors in the cell–cell interactions of SBOs and ACCs. The present study was designed to investigate MAPK signaling pathways in the hypertrophic changes of normal ACCs induced by OA SBOs using both direct and indirect coculture systems.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we demonstrated the importance of MAPK signaling pathways as the means by which OA subchondral bone osteoblasts induce altered phenotype changes in articular cartilage chondrocytes. Our experiments also provide some insight into the cross-talk taking place between the p38 and ERK-1/2 signaling pathways during this pathologic interaction process.
It was observed that ACCs isolated from OA patients produced significantly greater levels of mRNA for CBFA1, COL10, and ALP genes as compared with ACCs isolated from healthy patients. This finding indicates that the OA ACCs possessed greater potential to undergo hypertrophic differentiation; these results corroborate those of previous studies comparing normal and OA ACCs (28, 29).
Applying an in vitro indirect coculture model, Sanchez et al (12) demonstrated that sclerotic OA SBOs decreased cartilage-specific gene expression, such as SOX9 and COL2. They also showed that inhibitors of hypertrophic differentiation, such as parathyroid hormone–related protein and parathyroid hormone receptor were significantly down-regulated in ACCs cocultured with OA SBOs (12). These findings are evidence that OA SBOs can decrease the inhibitors of hypertrophic differentiation, leading to a subsequent mineralized matrix deposition in cartilage. In the present study, using both direct and indirect coculture methods, we showed that OA SBOs increased both hypertrophic gene expression and matrix mineralization.
Interestingly, hypertrophic changes are followed by a simultaneous decrease in chondrocyte-specific phenotype. A characteristic change of OA is an up-regulation of hypertrophy-related markers and mineralization-related markers (3) and a down-regulation of chondrocyte-specific markers (COL2 and AGG) in articular cartilage (30). The observations in our study suggest that the interaction of OA SBOs may lead to these typical hypertrophic changes in ACCs. It has been reported that the transition of ACCs to hypertrophic changes contributes to the activation of matrix metalloproteinases (MMPs), which precedes cartilage degeneration (31, 32), indicating that the phenotypic conversion of ACCs to hypertrophy is pathologic for the health and integrity of articular cartilage, leading to its degeneration.
The reasons why OA SBOs seem to induce the altered ACC phenotypes remain unclear; however, several potential pathways may be responsible. Both our own studies (data not shown) and those by other groups of investigators have demonstrated that OA SBOs produce abnormal levels of osteogenic markers, growth factors, and cytokines. Specifically, increased production of growth factors, such as insulin-like growth factor 1 (IGF-1) (33), and TGFβ (34), have been reported in OA SBOs. Among these factors, IGF-1 has been implicated in the induction of cartilage hypertrophic changes in growth plate chondrocytes (35, 36). In addition, it has been reported that OA SBOs produce abnormal levels of cytokines such as interleukin-1 and interleukin-6 (34), tumor necrosis factor, and MMP-13 (37, 38), all of which have the ability to activate a diverse array of signaling pathways in cartilage hypertrophy. It is therefore possible that the biomolecules secreted from OA SBOs communicate either individually or cooperatively with ACCs, thereby mediating the induction of phenotype changes of ACCs. Further studies are required to delineate the soluble factors from OA SBOs that are responsible for triggering the hypertrophic changes of ACCs in OA.
Among the signaling factors, the MAPK subtypes ERK-1/2 and p38 play a key role in the signaling process of chondrocyte cellular differentiation and homeostasis, depending on the nature of extracellular stimuli (14, 39). This knowledge prompted us to investigate MAPK signaling in the context of the influence of normal and OA SBOs on the differentiation of ACCs. This study is the first of its kind to show that OA SBOs induce ERK-1/2 phosphorylation and suppress p38 phosphorylation in ACCs, indicating that the alterations of these pathways accompany the pathologic phenotype changes in ACCs. Indeed, we have demonstrated that basal levels of ERK-1/2 phosphorylation increased and p38 decreased in OA ACCs as compared with normal ACCs, an indication of the pathologic relevance of these pathways in the pathogenesis of OA.
When the influence of ERK-1/2 phosphorylation was blocked by an inhibitor, p38 was activated in ACCs grown in the presence of OA SBO conditioned medium. The application of the ERK-1/2 inhibitor in OA SBO conditioned medium reversed ACC hypertrophy, and there was a return to the chondrogenic phenotype of ACCs. This observation implies that OA SBOs induced altered phenotype changes in ACCs via a deactivation of p38 and an activation of ERK-1/2 phosphorylation. This notion was further supported by results showing that when p38 was neutralized by an inhibitor in ACCs cocultured with normal SBOs, ERK-1/2 phosphorylation was augmented, and a weakening of chondrogenic gene expression and increase of hypertrophic gene expression was observed. Taken together, these data indicate that OA SBOs decrease p38 phosphorylation and increase ERK-1/2 activity, with a resulting reduction in chondrogenic phenotype and an increase in hypertrophic phenotype.
MAPKs are regulated at several levels, including kinase–kinase and kinase–substrate interactions and inhibition of cross-talk/output by the MAPKs themselves (40, 41). The activities of p38 are primarily governed by extensive cross-talk with ERK-1/2, a process that involves protein phosphatase, resulting in a reciprocal bidirectional equilibrium between ERK-1/2 and p38 phosphorylation, where an increase in p38 activity suppresses the activation of ERK-1/2 and vice versa (42). Such cross-talk appears to play a role in the OA SBO–regulated ACC phenotype, the existence of which has been shown in chondrocytes. For example, the opposing roles of ERK-1/2 and p38 have been demonstrated in chondrogenesis regulation (43). The finding that ERK-1/2 activation increased the hypertrophic differentiation of ACCs is consistent with study findings of a strong activation of the ERK-1/2 pathway in the hypertrophic zone of the growth plate (44). Furthermore, it has been demonstrated that the inhibition of ERK-1/2 delayed hypertrophic differentiation in growth plate chondrocytes during endochondral ossification (45).
It is possible that components of the p38 and ERK-1/2 pathways may interact directly in the transcription complex. The intermediate p38 and ERK-1/2 pathway substrates involved in these interaction are not known, but it is interesting that PD98059 (anti–ERK-1/2) significantly reduced the expression of the transcription factor CBFA1, whereas SB203580 (anti-p38) activated this transcription factor. During early skeletogenesis, chondrocyte hypertrophy is stimulated through the expression of CBFA1 in prehypertrophic chondrocytes, most likely by up-regulation of COL10 expression (46). Continuous expression of CBFA1 in chondrocytes induces hypertrophic differentiation and endochondral ossification, which is suggestive of an important role of this transcription factor in triggering hypertrophic changes (47). It is therefore possible that OA SBO–induced altered phenotype changes are triggered in ACCs via the activation of the MAPK/CBFA1 pathway.
In conclusion, this study demonstrated that OA SBOs could induce the activation of ERK-1/2 and the deactivation of p38 in articular cartilage chondrocytes, resulting hypertrophic changes of the chondrocytes. These data provide insight into the MAPK signaling pathways involved in the molecular mechanisms of the pathogenesis of OA, which may have significant clinical implications.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Xiao 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. Prasadam, Crawford, Xiao.
Acquisition of data. Prasadam, van Gennip, Friis, Shi, Crawford, Xiao.
Analysis and interpretation of data. Prasadam, van Gennip, Friis, Shi, Crawford, Xiao.