Osteoarthritis (OA) is one of the most prevalent chronic joint diseases and is characterized by progressive cartilage destruction and insufficient extracellular matrix synthesis. Thus far, none of the strategies employed to prevent and treat OA have been effective. The final option for advanced OA is the surgical approach of total joint replacement (1). Studies have shown that changes in growth factor signalings and their downstream target genes may be involved in the development of OA (2–5). However, the pathogenic mechanisms of OA are still largely unknown. Better understanding of the molecular events in OA development would provide important information that could facilitate the identification of novel therapeutic targets for the prevention and treatment of OA.
Chondrocytes are the only cell type in adult human articular cartilage in which the main responsibility is the synthesis and degradation of extracellular matrix (6). Numerous molecules and pathways, such as hypoxia-inducible factor 2α, discoidin domain receptor 2, and hedgehog signaling, in the articular chondrocytes have been shown to be involved in cartilage metabolism (7–11). Recently, the role of fibroblast growth factor (FGF) family members in the regulation of cartilage homeostasis has received specific attention (12). FGF-18 is a well-established anabolic growth factor that contributes to the metabolism of articular cartilage (13, 14). Although several lines of evidence support the tight association between FGF-2 and articular cartilage metabolism, the role of FGF-2 in cartilage homeostasis is controversial (12, 15).
Some studies of human articular chondrocytes have demonstrated that FGF-2 stimulates the production of matrix metalloproteinase 13 (MMP-13), which is the main collagenase responsible for collagen degradation (16). Results of other studies have suggested that FGF-2 functions as a chondroprotective factor in cartilage homeostasis (17–19). It has been reported that FGF-2 can suppress interleukin-1 (IL-1)–induced catabolic effects on human cartilage (18). In mice with Fgf2 deficiency, spontaneous OA, as well as instability-induced OA, is accelerated (19). In response to tissue injury or mechanical compression, FGF-2 can be released from the extracellular matrix to activate intracellular ERK signaling and regulate expression of chondrocyte-specific genes, suggesting that FGF-2 plays a homeostatic role in articular chondrocytes (20).
The involvement of FGF ligands in the maintenance of articular cartilage suggests that FGF receptors (FGFRs) may also play an important role in articular cartilage homeostasis. Notably, FGFR-1 and FGFR-3 are highly expressed in human articular chondrocytes and have been implicated in cartilage metabolism (21). Valverde-Franco et al showed that Fgfr3-knockout (KO) mice exhibited abnormal cartilage metabolism and early signs of OA, suggesting that FGFR-3 signaling may have a chondroprotective role during the development of OA (22). It is hypothesized that activation of FGFR-1 may exert antianabolic and procatabolic effects on adult human articular cartilage (12). However, since conditional deletion of Fgfr1 is known to have a lethal effect on mouse embryos (23), it is impossible to investigate the in vivo function of FGFR-1 in articular cartilage homeostasis using conventional Fgfr1-KO mice.
In this study, we investigated the role of FGFR-1 in articular cartilage function postnatally, using mice with tamoxifen-inducible and cartilage-specific conditional knockout (cKO) of the Fgfr1 gene (hereinafter referred to as Fgfr1 cKO mice). We evaluated the effects of FGFR-1 on the function of articular chondrocytes and the degeneration of articular cartilage in mice using 2 models of OA, as well as an antigen-induced arthritis (AIA) model. Our study demonstrated that Fgfr1 deficiency in mice attenuates articular cartilage degeneration in all 3 arthritis models. In addition, we found that blockade of FGFR-1 signaling could antagonize the IL-1β–induced up-regulation of MMP-13 and enhance the expression of FGFR-3 in human articular chondrocytes.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
OA is one of the most prevalent aging-related joint diseases and is the leading cause of disability in the elderly. Currently, there is no effective way to prevent and treat cartilage destruction in OA, which is a reflection of the insufficient understanding of the molecular mechanisms of the initiation and progression of OA. In our study, we provide evidence, for the first time, to indicate that genetic inhibition of Fgfr1 in mature adult mouse articular cartilage attenuated the development of OA.
Understanding of the physiologic role of FGFR-1 in the maintenance of articular cartilage homeostasis in vivo has been elusive, due to the lethal effects of conventional Fgfr1 knockout on mouse embryos (32). Adult mice with conditional knockout of the Fgfr1 gene in chondrocytes (Col2-Cre;Fgfr1fl/fl mice) will remain alive, but these mice undergo significant skeletal changes, including an increased height of the hypertrophic chondrocyte zone and significantly disturbed bone homeostasis (33), which may cause intrinsic changes in the articular cartilage and affect the susceptibility of articular cartilage to OA at the adult stage.
To determine the specific function of FGFR-1 in articular chondrocytes, we specifically disrupted Fgfr1 gene expression in articular cartilage of mice at an adult stage, in a tamoxifen-inducible and chondrocyte-specific manner, by crossing Fgfr1-floxed mice with Col2-CreERT2–transgenic mice. Fgfr1 cKO mice and their Cre-negative WT littermates were administered tamoxifen at the age of 8 weeks. Mice with the Fgfr1 deletion exhibited no gross abnormalities of body size, body weight, skeletal structure, or histologic features when compared to WT mice. Thus, these Fgfr1-deficient mice with grossly normal skeletons, joint structures, and morphologic features were suitable for study of OA at an adult stage.
To investigate the biologic effect of FGFR-1 on cartilage degeneration, 3 different arthritis models, including aging-associated spontaneous OA, surgery-induced OA, and AIA, were used. Histologic examination of the knee joints of mice in these 3 models revealed that deletion of the Fgfr1 gene resulted in a substantial protection against loss of aggrecan and against articular cartilage structural damage in the knee joint, demonstrating that down-regulation of FGFR-1 in articular cartilage has a chondroprotective effect, thereby slowing the development of OA. These observations are consistent with those from a recent in vitro study showing that FGFR-1 mainly transmits an FGF-2–mediated catabolic signal in human articular chondrocytes (21).
The primary characteristic of OA is an imbalance between anabolic effects on extracellular matrix synthesis and catabolic effects on matrix degradation. These dual processes of reduced matrix synthesis and/or increased production of degradative proteinases will lead to generation of articular cartilage matrix that is unable to transmit normal mechanical stress. The proteolytic functions of the ADAMTS and MMP family members play important roles in the development of OA. Adamts5, a key member in the ADAMTS family, is responsible for aggrecan degradation in mouse models of OA (34, 35), a feature that constitutes an early event in the development of OA (36). Furthermore, MMP-13 is considered the most active collagenase for type II collagen degradation, and greatly contributes to OA development (37). Genetically modified mice with constitutionally active MMP-13 expression showed development of OA, and Mmp13 deficiency protected mice against OA cartilage damage (38, 39).
FGF-2 has been shown to stimulate production of MMP-13 by activating the NF-κB and MAPK pathways in human articular chondrocytes (40). Moreover, IL-1β could stimulate the synthesis and secretion of multiple degradative enzymes (such as MMP-13) in cartilage, which contributes to OA development (41). In the present study, we found that deletion of the Fgfr1 gene decreased MMP-13 expression and significantly reversed the IL-1β–induced up-regulation of MMP-13 in primary chondrocytes. Consistent with this finding, we also found that inhibition of FGFR-1 with the selective inhibitor PD166866 antagonized the IL-1β–induced up-regulation of MMP-13 in human articular chondrocytes. In contrast, we did not detect any significant changes in Adamts5 expression in Fgfr1-deficient mouse chondrocytes. In addition, we observed that disruption of the Fgfr1 gene decreased the expression of type X collagen, enhanced the expression of aggrecan, and increased the accumulation of proteoglycan in articular cartilage.
These changes may be at least partly responsible for the decelerated progression of cartilage degeneration in Fgfr1-deficient mice. Our results suggest that Fgfr1 deficiency suppresses the expression of MMP-13 in articular chondrocytes, indicating that FGFR-1, as a critical regulator of MMP-13 expression, may be used as a therapeutic target for OA treatment.
The FGF family plays a critical role in cartilage development. In growth plate cartilage, FGFR-3 is mainly expressed in the proliferating and prehypertrophic chondrocytes. FGFR-3 suppresses the proliferation and differentiation of chondrocytes, while FGFR-1 functions as a potent mitogenic stimulator (42, 43). However, few studies have explored the physiologic role of FGFR signaling in vivo in adult articular chondrocytes. It has been reported that FGFR-1 and FGFR-2 are the predominant receptors in healthy mature articular cartilage in mice (19). In contrast, the major FGFRs expressed in human articular chondrocytes are considered to be FGFR-1 and FGFR-3 (21).
During OA development, FGFR-3 expression is down-regulated in articular chondrocytes from patients with OA (21), and early development of OA in Fgfr3-KO mice could be attributed to an increase in MMP-13 expression (22). We also found that expression of FGFR-3 was decreased in the knee joints of mice with OA induced by DMM surgery. These results suggest that down-regulation of FGFR-3 is at least partially responsible for the development of OA, and that FGFR-3 plays a potentially chondroprotective role in the articular cartilage.
FGFR-3–transmitted signaling in articular chondrocytes may have an effect on cartilage metabolism that is opposite to that of FGFR-1. In this study, we found that the absence of FGFR-1 in mouse articular cartilage not only could antagonize up-regulation of MMP-13 but also could lead to increased expression of FGFR-3 in articular chondrocytes. These results indicate that deletion of the Fgfr1 gene may, in addition to antagonizing the catabolic action of certain proteinases such as MMP-13 on cartilage, also delay the progression of OA by elevating the expression of FGFR-3. It is conceivable that knockdown of Fgfr3 in articular chondrocytes would attenuate the chondroprotective phenotype in Fgfr1-deficient mice. Studies involving chondrocyte-specific Fgfr1/Fgfr3 double-KO mice would provide further solid evidence to support the opposing roles of FGFR-1 and FGFR-3 in the development and progression of OA; these studies are now under way.
Overexpression of FGFR-3 could inhibit the Indian hedgehog (IHH) signaling pathway in the growth plate, and inhibition of IHH signaling is able to attenuate the severity of OA (11, 44). Whether IHH is involved in the chondroprotective effects of FGFR-3 requires further investigation. In addition, it has been reported that FGFR-3 is down-regulated by FGF-2 via the FGFR-1/ERK/MAPK pathway in human articular chondrocytes (21). Whether deletion of FGFR-1 in chondrocytes leads to up-regulation of FGFR-3 via down-regulation of the ERK/MAPK pathway needs further study.
In summary, these results show, for the first time, that conditional deletion of the Fgfr1 gene in mature mouse articular chondrocytes protects the knee joint cartilage against destruction, probably by suppressing the production of MMP-13 and enhancing the expression of FGFR-3. Our study provides valuable insights into the potential roles of FGFR-1 in articular cartilage metabolism. Therefore, these findings suggest that targeting of FGFR-1 could be a potential therapeutic strategy in patients with OA.
- Top of page
- 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. L. Chen 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. Weng, D. Chen, L. Chen.
Acquisition of data. Weng, Yi, Huang, Luo, Wen, Du, D. Chen, L. Chen.
Analysis and interpretation of data. Weng, Q. Chen, Deng, D. Chen, L. Chen.