Osteoarthritis (OA) is the most common musculoskeletal disease and is caused by age- or trauma-related changes in the homeostatic balance between anabolic and catabolic mechanisms (). The main pathogenetic mechanisms in OA are cartilage degradation induced by excessive mechanical stress, age-related changes in cells and extracellular matrix (ECM) mediated by the production of ECM-degrading enzymes, and inflammatory cytokines ([2, 3]). While articular cartilage damage is central to the OA process, this process also involves all other joint tissues. The anterior cruciate ligament (ACL) is critical to the biomechanical stability and function of the knee joint. Traumatic ACL injury can lead to cartilage damage and OA. Aging-associated OA also leads to structural changes in the ACL, which can contribute to disease progression ([4, 5]). In this regard, we recently reported that the cartilage degradation and ACL degeneration in OA knee joints show parallel progression (), suggesting the importance of molecular mechanisms of ACL degradation and regeneration in OA pathogenesis.
The ECM in the ACL consists predominantly of type I collagen, with small amounts of other collagens, proteoglycans, and other glycoproteins, including aggrecan, decorin, tenascins, and fibromodulin ([7, 8]). Collagen fibers provide tensile strength, and proteoglycans provide resistance to compression stress. ACL ruptures usually occur in the mid-substance of the femoral side ().
Cell populations in the ACL are generally referred to as fibroblast-like cells, although they are heterogeneous and also include subsets of progenitor cells ([9, 10]). Degraded ACL is characterized by changes in cell organization, cell death, and proliferation and by abnormal differentiation, most notably chondrocyte-like cell morphology and gene expression ([6, 11]). Understanding of the mechanisms that govern survival, differentiation, and ECM production by ligament cells is essential to developing new concepts for pathogenesis and therapeutic approaches.
Mohawk (MKX) and scleraxis (SCX) are the 2 transcription factors with relative specificity for tendon/ligament. SCX is a helix-loop-helix transcription factor that regulates the differentiation of tendon/ligament progenitors during skeletal development ([12, 13]). However, its expression level is low in mature ligament and tendon cells, suggesting that it may not play a major role in mature tendon/ligament homeostasis. MKX is also important during tendon and ligament development ([14-16]). Mkx-deficient mice have hypoplastic tendons throughout the body and deficient type I collagen production in tendon cells ([14, 16]). Importantly, we have found that Mkx expression is maintained in mature tendon/ligament cells in mice (), suggesting that it has a potential role in tendon/ligament tissue homeostasis and regeneration.
Based on these observations, we undertook the present investigation. We examined the expression patterns and function of MKX in human adult ligament and tendon tissue, in the context of OA pathogenesis.
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Mkx has recently been identified as a tendon/ligament specific transcription factor regulating expression of tendon/ligament ECM genes, including those for α1 chain of type I collagen, tenomodulin, and/or fibromodulin, during skeletal development in mice ([14, 16]); however, MKX expression and function in human tendon/ligament cells have not been elucidated. In this study, we demonstrated that MKX is expressed in adult human ligaments and its expression is clearly decreased in ligaments from patients with OA, correlating deficient expression of important ECM genes such as COL1A1 and TNXB.
The relationship between ACL degeneration and OA has been examined in earlier studies ([6, 26-28]). Typically, degenerative changes in the ACL start with collagen fiber disorientation, followed by mucoid degeneration, inflammatory cell infiltration, and/or neovascularization. IL-1β is among the critical proinflammatory cytokines involved in cartilage degradation during OA pathogenesis, as well as in ACL degeneration ([29, 30]). IL-1β stimulation in human tendon–derived cells significantly reduces expression of the genes for types I and III collagen, tenomodulin, and SCX and promotes expression of genes associated with ACL degeneration, such as those for aggrecanase, cyclooxygenase 2, matrix metalloproteinases 1, 3, and 13, ADAMTS-4, and IL-6 ([31, 32]). Herein we have shown that MKX is also down-regulated by IL-1β stimulation in human ligament cells. In addition, the down-regulation of MKX by siRNA in human ACL–derived cells reduces the expression of TNXB. These results support the notion that down-regulation of MKX is involved in ligament degeneration in OA and that MKX maintains ligament function and prevents degeneration.
As previously reported ([14, 16]), Mkx regulates type I collagen during tendon/ligament development in mice. In the present study, MKX knockdown in human ACL–derived cells did not significantly reduce the expression of COL1A1 messenger RNA (mRNA). However, Western blot analysis indicated that type I collagen protein expression was reduced by siMKX treatment. This discrepancy between mRNA and protein expression could be caused by an indirect effect of MKX knockdown via reduced TNXB expression. Tenascin-XB is a member of the tenascin family of ECM glycoproteins that contribute to matrix structure and regulate collagen fibrillogenesis via direct binding with type I collagen (). Studies of cultured dermal fibroblasts showed that fibroblasts from Tnxb−/− mice failed to deposit type I collagen into cell-associated matrix, although type I collagen synthesis by cells from Tnxb−/− and wild-type mice was similar ().
Interestingly, expression of the chondrogenic transcription factor SOX9 in human ACL–derived cells was increased with both IL-1β stimulation and MKX siRNA knockdown. Further, immunohistochemical analysis of human ACL tissue demonstrated that SOX9-positive cells were increased in ACLs from OA knees compared with normal ACLs. In this regard, we and others have previously demonstrated chondroid metaplasia in ACLs from knees with cartilage degeneration but not in ACLs with normal cartilage ([6, 10, 35]). Several reports have indicated that ACL-derived cells have high chondrogenic capacity and that SOX9 drives the differentiation from tenocyte to chondrocyte ([10, 36]).
It thus appears that ligament/tendon may contain a substantial proportion of progenitor cells. In OA, progenitor cells are activated, abnormally expressing SOX9 and differentiating to chondrocyte-like cells ([28, 37]). Although these abnormal cells in degenerated ACLs are frequently positive for SOX9, other chondrogenic markers such as type II collagen or aggrecanase are not expressed by all of these cells. In a recent study, we demonstrated that type II collagen or aggrecanase was present only in a subset of cells, even in areas with chondroid metaplasia (). This is consistent with our in vitro observations in the present study, in which increased SOX9 expression was not associated with increased expression of other chondrogenic markers such as COL2A1 and ACAN. Moreover, other studies on chondrogenesis of human mesenchymal stem cells have indicated that the increasing SOX9 expression precedes expression COL2A1 or ACAN ().
In this study we also sought to distinguish between changes that are related to normal aging and OA-related changes. We studied ACLs from donors age ≥60 years with no history of OA and minimal articular cartilage changes seen on macroscopic examination of the knees. The mean age of the normal aging group was similar to that of the OA group. We found that MKX levels differed between the 2 non-OA groups, with significantly lower levels in the aging group. Thus, there is an evident effect of aging on suppression of MKX. MKX concentrations were even lower in ACLs from OA-affected knees, indicating that OA-related mechanisms also contribute to the changes in MKX. The observation that IL-1β suppressed MKX in ACL-derived cells suggests a potential mechanism for this.
In conclusion, this study demonstrates that MKX is expressed in cells from normal adult human ACLs and that its expression is reduced in ACLs from OA-affected joints. In vitro, IL-1β suppresses MKX and enhances SOX9. These observations support the novel concept that loss of MKX, driven in part by proinflammatory cytokines such as IL-1β, leads to abnormal chondrocyte-like differentiation of ligament cells and production of ECM with deficient biomechanical properties (Figure 5). Correction of abnormal MKX expression could be pursued as a new approach to address tissue repair after injury and during chronic processes such as OA.
Figure 5. Hypothesis on the role of MKX in ligament homeostasis and degeneration. MKX maintains ligament function and prevents degeneration via regulation of TNXB and SOX9. Reduced expression of MKX in degenerated ligaments, mediated by proinflammatory cytokines, leads to abnormal differentiation and extracellular matrix production.
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- MATERIALS 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. Asahara 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. Nakahara, Ozaki, Lotz, Asahara.
Acquisition of data. Nakahara, Hasegawa, Otabe, Ayabe, Matsukawa.
Analysis and interpretation of data. Nakahara, Hasegawa, Otabe, Ayabe, Matsukawa, Onizuka, Ito, Lotz, Asahara.