ARTHRITIS & RHEUMATOLOGY Vol. 68, No. 10, October 2016, pp 2432–2443 DOI 10.1002/art.39739 C 2016, American College of Rheumatology V
Fibroblast Growth Factor Receptor 3 Inhibits Osteoarthritis Progression in the Knee Joints of Adult Mice Junzhou Tang, Nan Su, Siru Zhou, Yangli Xie, Junlan Huang, Xuan Wen, Zuqiang Wang, Quan Wang, Wei Xu, Xiaolan Du, Hangang Chen, and Lin Chen Objective. Fibroblast growth factor (FGF) signaling is involved in articular cartilage homeostasis. This study was undertaken to investigate the role and mechanisms of FGF receptor 3 (FGFR-3) in the pathogenesis of osteoarthritis (OA) caused by surgery and aging in mice. Methods. FGFR-3 was conditionally deleted or activated in articular chondrocytes in adult mice subjected to surgical destabilization of the medial meniscus (DMM). A mouse model of human achondroplasia was also used to assess the role of FGFR-3 in age-associated spontaneous OA. Knee joint cartilage was histologically evaluated and scored using the Osteoarthritis Research Society International system. The expression of genes associated with articular cartilage maintenance was quantitatively evaluated in hip cartilage explants. The effect of inhibiting Indian hedgehog (IHH) signaling in Fgfr3-deficient explants was analyzed. Results. Conditional Fgfr3 deletion in mice aggravated DMM-induced cartilage degeneration. Matrix metalloproteinase 13 and type X collagen levels were
up-regulated, while type II collagen levels were downregulated, in the articular cartilage of these mice. Conversely, FGFR-3 activation attenuated cartilage degeneration induced by DMM surgery and age. IHH signaling and runt-related transcription factor 2 levels in mouse articular chondrocytes were up-regulated in the absence of Fgfr3, while inhibition of IHH signaling suppressed the increases in the expression of Runx2, Mmp13, and other factors in Fgfr3-deficient mouse cartilage explants. Conclusion. Our findings indicate that FGFR-3 delays OA progression in mouse knee joints at least in part via down-regulation of IHH signaling in articular chondrocytes. Osteoarthritis (OA) is a common degenerative joint disease involving progressive cartilage degeneration that eventually leads to disability. There are few strategies for preventing or treating OA (1). Understanding the underlying mechanisms can facilitate the development of novel therapies. Various molecules and pathways involved in cartilage or joint development are implicated in OA pathogenesis (2,3) and could potentially serve as therapeutic targets. The fibroblast growth factor (FGF) signaling pathway plays important roles in the regulation of skeletal development (4). FGFs exert their effects by binding to FGF receptors (FGFRs) (5), which are expressed in specific spatiotemporal patterns in the developing and adult skeletons. For example, FGFR-3 is initially expressed by chondrocytes located in the central core of mesenchymal condensation during early development, then in proliferating and prehypertrophic zones of growth plates and articular chondrocytes (6). Mutations in FGFR3 have been linked to a variety of human genetic skeletal dysplasias. Gain-of-function mutations in FGFR3 result in chondrodysplasias, including achondroplasia, hypochondroplasia, and thanatophoric dysplasia, while
Supported by the Special Funds for Major State Basic Research Program of China (973 Program) (grant 2014CB942904), the National Natural Science Foundation of China (grants 81530071 and 81220108020), the Committee of Science and Technology of Chongqing (grant CSTC 2011jjA1468), and Program for Innovation Team Building at Institutions of Higher Education in Chongqing (grant CXTDG201602019). Junzhou Tang, MD, Nan Su, MD, Siru Zhou, MD, Yangli Xie, MD, Junlan Huang, BS, Xuan Wen, MD, Zuqiang Wang, MD, Quan Wang, MD, Wei Xu, MD, Xiaolan Du, MS, Hangang Chen, BS, Lin Chen, MD, PhD: Center of Bone Metabolism and Repair, State Key Laboratory of Trauma, Burns, and Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, China. Drs. Tang, Su, and Zhou contributed equally to this work. Address correspondence to Lin Chen, MD, PhD, or Yangli Xie, MD, Department of Rehabilitation Medicine, Center of Bone Metabolism and Repair, State Key Laboratory of Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China. E-mail:
[email protected] or
[email protected]. Submitted for publication July 16, 2015; accepted in revised form April 26, 2016. 2432
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loss-of-function mutations in FGFR3 lead to camptodactyly, tall stature, scoliosis, and hearing loss syndrome in humans (7,8). Similar skeletal phenotypes have also been observed in mice with corresponding genetic modifications in Fgfr3 (7). Thus, FGFR-3 is a negative regulator of endochondral bone development. The important role of FGFR-3 in skeletal development and human genetic skeletal dysplasias indicates that FGFR-3 may play an essential role in the maintenance of articular cartilage, which is supported by several studies. FGFR-3 is abundantly expressed in articular chondrocytes in adulthood (9 2 11) and has been shown to be down-regulated in OA patients (10,11). Valverde-Franco et al (12) showed that conventional FGFR-32knockout (FGFR-32/2) mice develop early-onset arthritis. Moreover, severe bowleg and a higher incidence of obesity, which are well-known risk factors for OA, are observed in patients with achondroplasia, but OA rarely develops (13). These findings imply that FGFR-3 prevents articular cartilage degeneration. However, because of the intrinsic disadvantages of FGFR-32/2 mice and achondroplasia patients described below, the true effects of FGFR-3, especially the direct effect of FGFR-3 on the maintenance of articular cartilage homeostasis in adulthood, are not clear. Many factors, such as embryonic and early skeletal development status, are involved in the pathogenesis of OA (14). Additionally, although articular cartilage is the major tissue affected in OA, all joint components, including the subchondral bone and synovium, are involved in the pathogenesis of this disease (15,16). FGFR-32/2 mice and patients with achondroplasia have mutant FGFR3 genes in all cells/tissues and exhibit abnormal skeletal development and dysregulated bone quality (17–19), making it difficult to exclude the role of tissues other than articular cartilage in OA pathogenesis. More evidence to convincingly demonstrate the direct role of FGFR-3 in articular cartilage maintenance and its underlying mechanisms are needed. To circumvent this issue and investigate the role of FGFR-3 in OA pathogenesis, we induced conditional Fgfr3 deletion or activation in the chondrocytes of adult mice. We found that FGFR-3 directly protects adult mouse articular cartilage against degeneration by inhibiting chondrocyte hypertrophy and maintaining the levels of extracellular matrix (ECM) proteins, mainly through inhibition of Indian hedgehog (IHH)/ runt-related transcription factor (RUNX-2) signaling. MATERIALS AND METHODS Mouse model of OA. Fgfr3flox/flox (Fgfr3fl/fl) mice were generated in our laboratory (20). Fgfr3G369C/1 (21), Fgfr3K644E/neo
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(22), and Col2a1-CreERT2 (23) mice were provided by Drs. Chuxia Deng (University of Macau, Macau, China) and Di Chen (Rush University, Chicago, IL). Fgfr3fl/fl mice were crossed with Fgfr3fl/fl;Col2a1-CreERT2 mice to obtain Fgfr3fl/fl;Col2a1-CreERT2 mice (FGFR-32conditional-knockout mice), and Fgfr3fl/fl mice (Cre-negative control mice). Fgfr3K644E/neo mice with an activating Fgfr3 mutation (K644E) were crossed with Fgfr3K644E/neo;Col2a1CreERT2 mice to obtain Fgfr3K644E/neo;Col2a1-CreERT2 mice (FGFR-32conditional-activation mice) and Fgfr3K644E/neo mice (Cre-negative control mice). Fgfr3G369C/1 mice were mated with Fgfr31/1 mice to generate Fgfr3G369C/1 mice (Fgfr3ACH/1 mice [with achondroplasia]) and Fgfr31/1 (wild-type) mice. Male FGFR-32conditional-knockout and FGFR-32conditionalactivation mice and their Cre-negative littermates were injected intraperitoneally with tamoxifen (Sigma-Aldrich) at 2 months of age (1 mg/10 gm/day for 5 days) before surgical induction of OA (9) by destabilization of the medial meniscus (DMM) in the right knee joint (24). As a control, sham operation was performed on the left knee joint with medial capsulotomy only. Mice with achondroplasia ages 6, 12, and 20 months were used to study the role of FGFR-3 in age-associated spontaneous OA. All animal protocols were approved by the Institutional Animal Care and Use Committee of Daping Hospital. Specimen preparation. Mice were killed and knee joints were fixed in 4% paraformaldehyde, decalcified in 20% formic acid, and embedded in paraffin. Serial sagittal sections were obtained across the entire joint by collecting 5-mm sections at 50-mm intervals. Sections were stained with Safranin O2fast green for histologic analysis. The intensity of Safranin O staining of growth plates in the femora and tibiae was used as an internal control between batches. The intervening sections were used for immunohistochemical analysis. Microscopy. Histologic changes in the medial tibial plateau and medial femoral condyle of knee joints were scored on a scale of 0 2 6 according to the recommendations of the Osteoarthritis Research Society International (OARSI) (25). The maximum scores for the medial femora and medial tibiae were calculated separately, and the summed scores (i.e., the sum of the 4 highest scores for each specimen) were used to evaluate the severity of cartilage destruction (25). Scoring was carried out by 3 independent investigators (JT, SZ, and YX). Immunohistochemical analysis. Immunohistochemical analysis was carried out using SP-9000 Histostain-Plus kits (Zsgb Bio). Briefly, endogenous peroxidase activity was quenched with 3% H2O2, followed by antigen retrieval with 0.1% trypsin and blocking with normal goat serum for 30 minutes. Sections were incubated overnight at 48C with antibodies against the following proteins: FGFR-3 (1:100; Santa Cruz Biotechnology), type II collagen (1:100; Chondrex), type X collagen (1:400; Abcam), matrix metalloproteinase 13 (MMP-13) (1:300; Proteintech), aggrecan neopeptide (1:200; Millipore), and RUNX-2 (1:200; Santa Cruz Biotechnology). After rinsing with phosphate buffered saline (PBS), sections were incubated with appropriate biotinylated secondary antibody and horseradish peroxidase2conjugated streptavidin2biotin. Immunoreactivity was visualized with a 3,30 diaminobenzidine tetrahydrochloride kit (Zsgb Bio) followed by counterstaining with methyl green. The numbers of positive cells in 3 central regions of articular cartilage were counted using Image Pro Plus version 5.1 software (Media Cybernetics).
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Figure 1. Histologic analysis of structural damage in the articular cartilage of FGFR-3–conditional-knockout mice (Fgfr3 cKO). A–D, Representative images of Safranin O–fast green–stained sections of knee joints from Cre-negative control mice (A and C) and FGFR-3–conditional-knockout mice (B and D) 1 month after sham operation, showing increased numbers of hypertrophic chondrocytes (arrowheads) and loss of extracellular matrix (ECM) in the articular cartilage of mutant mice. C and D show higher-magnification views of the boxed areas in A and B, respectively. E and F, Representative images of structural damage in articular cartilage from Cre-negative mice (E) and FGFR-3–conditional-knockout mice (F) 1 month after surgical destabilization of the medial meniscus (DMM). G–J, Severity of articular cartilage damage in Cre-negative and FGFR-3–conditional-knockout mice 1 month after sham operation or DMM surgery, evaluated using the Osteoarthritis Research Society International (OARSI) scoring system. Total (sum) scores (G and H) and maximal scores (I and J) were calculated for the medial femoral condyle (MFC) (G and I) and medial tibial plateau (MTP) (H and J). K– N, Representative images of Safranin O–fast green–stained sections of knee joints from Cre-negative mice (K and M) and FGFR-3–conditionalknockout mice (L and N) 2 months after sham operation, showing increased numbers of hypertrophic chondrocytes (arrowheads) and loss of ECM in the articular cartilage of mutant mice. M and N show higher-magnification views of the boxed areas in K and L, respectively. O and P, Representative images of structural damage in articular cartilage from Cre-negative mice (O) and FGFR-3–conditional-knockout mice (P) 2 months after DMM surgery. Q–T, Severity of articular cartilage damage in Cre-negative and FGFR-3–conditional-knockout mice 2 months after sham operation or DMM surgery, evaluated using the OARSI scoring system. Total scores (Q and R) and maximal scores (S and T) were calculated for the medial femoral condyle (Q and S) and medial tibial plateau (R and T). Bar 5 200 mm in A; 50 mm in C. Images in E, F, O, and P show the same magnification view as A. In G–J and Q–T, symbols indicate individual mice; horizontal lines and error bars show the mean 6 SD (n 5 14216 mice per group). * 5 P , 0.05; ** 5 P , 0.01. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39739/abstract.
Isolation and culture of articular cartilage explants and IHH signaling inhibitor treatment. Articular cartilage explants were isolated from femoral head cartilage of 425-
week-old FGFR-32conditional-knockout mice and their Crenegative littermates. Explants were washed in sterile PBS and cultured in Dulbecco’s modified Eagle’s medium/F-12 (1:1)
FGFR-3 PROTECTS ARTICULAR CARTILAGE AGAINST DEGENERATION
supplemented with 10% fetal bovine serum and penicillin/ streptomycin in a CO2 incubator at 378C for 48 hours. The explants were treated with 1 mM 4-hydroxytamoxifen (SigmaAldrich) for 72 hours to delete Fgfr3 in chondrocytes by Cremediated recombination, with Cre-negative explants serving as controls. Cartilage explants were then treated with 1 mM smoothened inhibitor (GDC-0449; Selleck Chemicals) or vehicle (dimethyl sulfoxide; Sigma-Aldrich) for 24 hours. Real-time polymerase chain reaction (PCR). Cartilage explants were flash-frozen for RNA extraction using TRIzol reagent (Invitrogen) and used to generate a complementary DNA template for real-time PCR, which was carried out on an Mx3000P system (Stratagene) using a SYBR Green RT-PCR kit (Takara Bio). Samples were prepared in quadruplicate, and the following forward and reverse primers were used: for cyclophilin A (internal control), 50 -CGA-GCT-CTG-AGCACT-GGA-GA-30 (forward) and 50 -TGG-CGT-GTA-AAGTCA-CCA-CC-30 (reverse); for Ihh, 50 -TGG-GAC-ACT-TGTGGT-GGA-GGA-30 (forward) and 50 -AGG-CGG-TAGAGC-ATC-TGA-GGG-30 (reverse); for Gli1, 50 -GGT-CCGGAT-GCC-CAC-GTG-AC-30 (forward) and 50 -TCC-CGCTTG-GGC-TCC-ACT-GT-30 (reverse); for Runx2, 50 -CCTGAA-CTC-TGC-ACC-AAG-TC-30 (forward) and 50 -GAGGTG-GCA-GTG-TCA-TCA-TC-30 (reverse); for Mmp13, 50 CAG-TTG-ACA-GGC-TCC-GAG-AA-30 (forward) and 50 (reverse); for CGT-GTG-CCA-GAA-GAC-CAG-AA-30 Adamts5, 50 -GGA-GCG-AGG-CCA-TTT-ACA-AC-30 (forward) and 50 -CGT-AGA-CAA-GGT-AGC-CCA-CTT-T-30 (reverse); for Col10a1, 50 -GCA-GCA-TTA-CGA-CCC-AAGAT-30 (forward) and 50 -CAT-GAT-TGC-ACT-CCC-TGAAG-30 (reverse); for Col2a1, 50 -CTG-GTG-GAG-CAG-CAAGAG-CAA-30 (forward) and 50 -CAG-TGG-ACA-GTA-GACGGA-GGA-AAG-30 (reverse); and for aggrecan, 50 -CACGCT-ACA-CCC-TGG-ACT-TTG-30 (forward) and 50 -CCATCT-CCT-CAG-CGA-AGC-AGT-30 (reverse). Statistical analysis. Data were analyzed using GraphPad Prism version 6.01. Results are presented as the mean 6 SD. Mean differences were evaluated by Student’s ttest or by two-way analysis of variance with a Bonferroni post hoc test. P values less than 0.05 were considered significant.
RESULTS Acceleration of articular cartilage destruction in adult mice with chondrocyte-specific deletion of Fgfr3. In adult mice with chondrocyte-specific deletion of Fgfr3, the skeleton was normal 1 and 2 months after tamoxifen injection (data not shown). A DMM model was used to assess the effects of Fgfr3 deficiency on OA development. Histologic analysis revealed a greater number of hypertrophic chondrocytes in the articular cartilage of FGFR-3–conditional-knockout mice as compared to Cre-negative mice 1 and 2 months after sham operation (Figures 1A–D and K–N). One month after DMM surgery, early signs of OA development, such as loss of cartilage and proteoglycan, were detected by Safranin O–fast green staining in FGFR-3– conditional-knockout mice; these phenotypes were
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more severe than those in Cre-negative mice (Figures 1E and F). Two months after DMM surgery, there was more extensive destruction of articular cartilage in most areas, with some exposure of the subchondral bone in FGFR-3–conditional-knockout mice as compared to Cre-negative mice (Figures 1O and P). We used the OARSI scoring system to quantify the severity of cartilage damage. The summed and maximal scores for the femora and tibiae were higher in FGFR-3–conditionalknockout mice than in Cre-negative mice at 1 and 2 months after DMM surgery (Figures 1G–J and Q–T). These results suggest that chondrocyte-specific deletion of Fgfr3 in adult mice exacerbates the OA phenotypes induced by DMM surgery. Disruption of articular cartilage homeostasis in FGFR-3–deficient mice. FGFR-3 protein expression was markedly reduced in articular chondrocytes of FGFR-3– conditional-knockout mice 1 month after tamoxifen injection (Figures 2A, B, and K), indicating that the Fgfr3 gene was effectively deleted. Chondrocyte hypertrophy plays an important role in endochondral ossification during skeletal development (26). In addition, chondrocyte hypertrophy is considered to be a positive factor for the initiation and progression of cartilage degeneration during OA development (27). FGFR-3 is abundantly expressed in the proliferative zone of growth plates and inhibits endochondral ossification by suppressing chondrocyte proliferation and hypertrophic differentiation (6). To clarify the mechanism of the acceleration of OA in FGFR-3–conditional-knockout mice, we assessed the levels of proteins related to chondrocyte hypertrophy by immunohistochemistry. Type X collagen and MMP-13, an important catabolic enzyme in cartilage ECM, are hypertrophic chondrocyte markers (27). Type II collagen was abundantly expressed in normal articular cartilage in Cre-negative mice (Figure 2C), whereas expression of type X collagen and MMP-13 was negligible (Figures 2E and G). In contrast, immunoreactivity of type X collagen (Figures 2E, F, and L) and MMP-13 (Figures 2G, H, and M) was increased, while that of type II collagen was decreased (Figures 2C and D) in articular cartilage from FGFR-3–conditional-knockout mice compared to Cre-negative mice. Moreover, the expression of aggrecan neopeptide, which reflects the cleavage of aggrecan by ECM enzymes (28), was increased in mutant mice relative to control mice (Figures 2I, J, and N), which was associated with a reduction in Safranin O staining of articular cartilage in mutant mice (Figures 1A–D). These results indicate that Fgfr3 deficiency directly promotes hypertrophy and enhances catabolic activity in articular chondrocytes, which may disrupt articular cartilage homeostasis in adult mice.
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Figure 2. Effects of Fgfr3 deletion on articular cartilage homeostasis in adult mice. Sections of knee joints from Cre-negative mice and FGFR32conditional-knockout mice (Fgfr3 cKO) were analyzed 1 month after tamoxifen injection. A and B, Detection of fibroblast growth factor receptor 3 (FGFR-3) expression in Cre-negative mice (A) and FGFR-32conditional-knockout mice (B) by immunohistochemistry. A decrease in the fraction of FGFR-32positive cells was observed in mutant mice. C and D, Detection of type II collagen expression in Cre-negative mice (C) and FGFR32conditional-knockout mice (D) by immunohistochemistry. A decrease in type II collagen expression was observed in mutant mice. E and F, Detection of type X collagen expression in Cre-negative mice (E) and FGFR-32conditional-knockout mice (F) by immunohistochemistry. G and H, Detection of matrix metalloproteinase 13 (MMP-13) expression in Cre-negative mice (G) and FGFR-32conditional-knockout mice (H) by immunohistochemistry. I and J, Detection of aggrecan neopeptide (aggrecan neo) expression in Cre-negative mice (I) and FGFR-32conditional-knockout mice (J) by immunohistochemistry. In A2J, broken lines indicate the chondro-osseous junction. Bar 5 50 mm. K2N, Percentages of cells positive for FGFR-3 (K), type X collagen (L), MMP-13 (M), and aggrecan neopeptide (N). Expression levels of type X collagen, MMP-13, and aggrecan neopeptide were increased in mutant mice. Symbols indicate individual slides; horizontal lines and error bars show the mean 6 SD (n 5 5 slides per genotype). * 5 P , 0.05; ** 5 P , 0.01. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39739/abstract.
Chondrocyte-specific FGFR-3 activation in adult mice delays the articular cartilage destruction induced by DMM surgery. Given the negative effects of FGFR-3 on chondrocyte hypertrophy, we speculated that FGFR-3 signaling plays a chondroprotective role in OA development. To test this hypothesis, we examined the phenotype of FGFR-3–conditional-activation mice after DMM surgery. These mice had grossly normal skeletons 1 and 2 months after tamoxifen injection (results not shown). In contrast to the increased number of hypertrophic chondrocytes in the articular cartilage of FGFR-3–conditional-knockout mice, FGFR-3–
conditional-activation mice were histologically comparable to Cre-negative mice 1 and 2 months after sham operation (Figures 3A, B, I, and J). After DMM surgery, FGFR-3–conditional-activation mice showed a milder OA phenotype than Cre-negative mice (Figures 3C, D, K, and L). The summed and maximal scores for femoral and tibial cartilage damage were lower in FGFR-3–conditional-activation mice than in Crenegative mice at 1 and 2 months after DMM surgery (Figures 3E–H and M–P), suggesting that OA progression induced by DMM was attenuated by FGFR-3 activation in chondrocytes.
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Figure 3. Histologic analysis of structural damage in the articular cartilage of FGFR-32conditional-activation mice (Fgfr3 cAct). A and B, Representative images of Safranin O2fast green2stained sections of knee joints from Cre-negative mice (A) and FGFR-32conditional-activation mice (B) 1 month after sham operation, showing no significant changes in articular cartilage. C and D, Representative images of structural damage in articular cartilage from Cre-negative mice (C) and FGFR-32conditional-activation mice (D) 1 month after DMM surgery. E2H, Severity of articular cartilage damage in Cre-negative mice and FGFR-32conditional-activation mice 1 month after sham operation or DMM surgery, evaluated using the OARSI scoring system. Total scores (E and F) and maximal scores (G and H) were calculated for the medial femoral condyle (E and G) and medial tibial plateau (F and H). I and J, Representative images of Safranin O2fast green2stained sections of knee joints from Cre-negative mice (I) and FGFR-32conditional-activation mice (J) 2 months after sham operation, showing no significant changes in articular cartilage. K and L, Representative images of structural damage in articular cartilage from Cre-negative mice (K) and FGFR-32conditional-activation mice (L) 2 months after DMM surgery. M2P, Severity of articular cartilage damage in Cre-negative mice and FGFR-32conditional-activation mice 2 months after sham operation or DMM surgery, evaluated using the OARSI scoring system. Total scores (M and N) and maximal scores (O and P) were calculated for the medial femoral condyle (M and O) and medial tibial plateau (N and P). Bar in A 5 200 mm. In E2H and M2P, symbols indicate individual mice; horizontal lines and error bars show the mean 6 SD (n 5 14215 mice per group). * 5 P , 0.05; ** 5 P , 0.01. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39739/abstract.
Delayed development of age-associated OA in mice harboring an Fgfr3 G369C mutation. Achondroplasia is caused by a gain-of-function mutation in FGFR3 and is the most common type of human genetic dwarfism (18). Interestingly, patients with achondroplasia develop severe bowleg deformity but rarely develop OA (13). To further
investigate this clinical phenomenon, we examined the articular cartilage phenotype of mice with the Fgfr3 G369C gain-of-function mutation (Fgfr3ACH/1 mice), which mimics human achondroplasia, in an age-associated spontaneous OA model. Fgfr3ACH/1 and wild-type mice showed grossly normal cartilage at 6 months of age (Figures 4A, B, and G–
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Figure 4. Histologic analysis of structural damage in the articular cartilage of Fgfr3ACH/1 mice. A–F, Representative images of Safranin O–fast green– stained sections of knee joints from wild-type (WT) mice (A, C, and E) and Fgfr3ACH/1 mice (B, D, and F) at 6 months of age (A and B), 12 months of age (C and D), and 20 months of age (E and F), showing less severe cartilage damage and ECM loss in mutant than in wild-type mice with advancing age. Bar 5 200 mm. G–J, Severity of articular cartilage damage in wild-type and Fgfr3ACH/1 mice, evaluated using the OARSI scoring system. Total scores (G and I) and maximal scores (H and J) were calculated for the medial femoral condyle (G and H) and medial tibial plateau (I and J). Symbols indicate individual mice; horizontal lines and error bars show the mean 6 SD (n 5 527 mice per group). * 5 P , 0.05. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39739/abstract.
J). Although stronger Safranin O staining was observed in Fgfr3ACH/1 mice at 12 months, there were no structural differences in the knee joints relative to wild-type mice (Figures 4C, D, and G–J). At the age of 20 months, wild-type mice showed OA phenotypes in the knee joint, including spontaneous cartilage erosion and loss of Safranin O staining (Figure 4E). In contrast, Fgfr3ACH/1 mice showed a comparatively mild phenotype (Figures 4E, F, and G–J). Suppression of articular cartilage chondrocyte hypertrophy in OA by FGFR-3 activation. To investigate the mechanism by which FGFR-3 activation in articular chondrocytes delays OA development, chondrocyte hypertrophy marker expression was analyzed by immunohistochemistry. Type X collagen and MMP-13 were abundantly expressed in the cartilage of Cre-negative mice 1 month after DMM; however, the levels were markedly reduced in FGFR-3–conditional-activation mice (Figures
5A–E). Fgfr3ACH/1 mice also showed decreased type X collagen and MMP-13 levels relative to wild-type mice at 20 months of age (Figures 5F–J). These data indicate that FGFR-3 activation protects articular cartilage against OA via suppression of chondrocyte hypertrophy. Increased IHH signaling and RUNX-2 expression mediate OA development caused by Fgfr3 deficiency. IHH signaling is important for normal endochondral ossification during bone development (29,30). In addition, IHH signaling is considered to be a mediator for the catabolic metabolism in OA cartilage destruction (31,32). We previously observed that growth plate chondrocytes of Fgfr3ACH/1 mice expressed lower levels of Ihh, suggesting that activation of FGFR-3 signaling inhibits IHH production in these cells (33). We therefore investigated whether IHH signaling mediates the aggravation of OA caused by Fgfr3 deficiency. IHH binds to the patched
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Figure 5. Effects of fibroblast growth factor receptor 3 (FGFR-3) activation on the homeostasis of articular cartilage in surgical destabilization of the medial meniscus (DMM)2induced and aging-associated osteoarthritis. A2D, Detection of type X collagen (A and B) and matrix metalloproteinase 13 (MMP-13) (C and D) expression in knee joint sections from 3-month-old Cre-negative and FGFR-32conditional-activation (Fgfr3 cAct) mice by immunohistochemistry. E, Percentages of type X collagen2positive cells and MMP-132positive cells in Cre-negative mice and mutant mice. F2I, Detection of type X collagen (F and G) and MMP-13 (H and I) expression in knee joint sections from 20-month-old wildtype (WT) and Fgfr3ACH/1 mice by immunohistochemistry. J, Percentages of type X collagen2positive and MMP-132positive cells in wild-type and mutant mice. In A2D and F2I, broken lines indicate the chondro-osseous junction. Bar 5 50 mm. In E and J, symbols indicate individual slides; horizontal lines and error bars show the mean 6 SD (n 5 5 slides per genotype). * 5 P , 0.05; ** 5 P , 0.01. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39739/abstract.
receptor, resulting in the dissociation of smoothened and activation of downstream effectors such as Gli transcription factors (34). We found that Ihh and Gli1 levels were up-regulated in Fgfr3-deficient cartilage explants from mouse hip joints (Figures 6A and B), suggesting that Fgfr3 deletion enhanced IHH signaling in articular chondrocytes. The transcription factor RUNX-2 also modulates chondrocyte hypertrophy as well as the expression of various ECM-degrading enzymes (31,35–37). We found that Runx2 levels were up-regulated in Fgfr3-deficient cartilage explants (Figure 6C), with similar results observed by immunohistochemistry in articular chondrocytes of FGFR-3–conditional-knockout mice 1 month after tamoxifen injection (Figures 6D–F). Since enhanced IHH signaling is a potential mechanism underlying the aggravated OA development observed in FGFR-3–conditional-knockout mice, we next investigated whether OA development could be delayed by inhibiting IHH signaling. GDC-0449 (38) is a selective inhibitor of smoothened, as evidenced by the reduction in Gli1 expression in mouse articular cartilage
explants (Figure 6G). Although IHH signaling was effectively inhibited, there was no change in Runx2, Mmp13, Adamts5, Col10a1, Col2a1, or aggrecan expression in Cre-negative mouse hip joint cartilage explants upon treatment with smoothened inhibitor (Figures 6H–M). Expression of Mmp13, Adamts5, and Col10a1 was up-regulated, while the expression of Col2a1 was down-regulated, in Fgfr3-deficient mouse cartilage explants (Figures 6I–L), indicating that chondrocyte hypertrophy and catabolism were induced. Furthermore, the expression of aggrecan was down-regulated in Fgfr3-deficient mouse cartilage explants (Figure 6M). Treatment with smoothened inhibitor for 24 hours abrogated the increase in Runx2, Mmp13, Adamts5, and Col10a1 expression in Fgfr3-deficient mouse explants (Figures 6I–K). However, no significant changes in Col2a1 or aggrecan messenger RNA levels were observed in Fgfr3-deficient mouse cartilage explants treated with smoothened inhibitor compared to those treated with vehicle (Figures 6L and M), indicating that FGFR-3–mediated anabolic effects on articular
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Figure 6. Fgfr3 deletion induces Indian hedgehog (IHH) signaling and runt-related transcription factor 2 (RUNX-2) expression. A2C, Realtime polymerase chain reaction (PCR) analysis of Ihh (A), Gli1 (B), and Runx2 (C) expression in cartilage explants isolated from the hip joints of Cre-negative and FGFR-32conditional-knockout mice (Fgfr3 cKO) after tamoxifen treatment. D and E, Detection of RUNX-2 expression in sections of knee joints from Cre-negative mice (D) and FGFR-32conditional-knockout mice (E) 1 month after tamoxifen injection. Broken line indicates the chondro-osseous junction. Bar 5 50 mm. F, Percentages of RUNX-22positive cells in Cre-negative and mutant mice. Symbols indicate individual slides; horizontal lines and error bars show the mean 6 SD (n 5 5 slides per genotype). G2M, Real-time PCR analysis of Gli1 (G), Runx2 (H), Mmp13 (I), Adamts5 (J), Col10a1 (K), Col2a1 (L), and aggrecan (M) gene expression in Cre-negative or Fgfr3-deficient mouse cartilage explants treated with vehicle or smoothened inhibitor (SMOi) GDC-0449. N, Schematic illustration of the mechanisms of FGFR-3 action in the maintenance of articular cartilage homeostasis in adult mice. FGFR-3 inhibits IHH signaling and promotes anabolic effects. IHH signaling induces chondrocyte hypertrophy and enhances the expression of extracellular matrix (ECM)2degrading enzymes, such as ADAMTS-5 and MMP-13, possibly via a RUNX-22dependent mechanism. In A2C and G2M, bars show the mean 6 SD (n 5 4 mice per genotype). * 5 P , 0.05; ** 5 P , 0.01.
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cartilage may be independent of IHH signaling. These results suggest that FGFR-3 regulates chondrocyte hypertrophy and catabolism in part via activation of IHH signaling (Figure 6N). DISCUSSION OA is a common disease in humans, characterized by progressive destruction of articular cartilage, which is difficult to regenerate naturally (39). Many molecules have been implicated in OA pathology, but the few strategies that target them have not proven effective for preventing or delaying OA progression (1). The important role of FGF signaling in OA pathogenesis has recently been identified (40). Several FGFs, including FGF-2, 8, and 18, have been shown to be negatively or positively involved in the pathogenesis of OA in animal and human studies. Multiple studies have demonstrated that FGF-18 protects articular cartilage against degeneration in adults (41,42). FGF-8 promotes cartilage degradation (43). FGF-2 has been shown to modulate articular cartilage metabolism, although these findings are a subject of controversy (10,44–48). Unfortunately, none of these factors have shown clear protective effects on articular cartilage in clinical trials, indicating that the effects and mechanism of FGFs on adult articular cartilage maintenance are complicated. FGF ligands exert their effect through FGFRs (5). The complex cross binding affinity among different FGFs and FGFRs, i.e., each individual FGF can bind and then activate several FGFRs in cartilage, may be responsible for the varying effects of FGFs on articular cartilage (40,45). Therapies targeting receptors appear to be more specific and are attracting increasing attention (49). FGF-2, 8, and 18 can bind to FGFR-3 with relatively high affinity (5), indicating that targeting FGFR-3 in articular cartilage is a good strategy for OA therapy if we accurately understand the role of FGFR-3 in the homeostasis of adult articular cartilage. We investigated the direct effect of FGFR-3 on adult articular cartilage maintenance using mice in which Fgfr3 was deleted or activated specifically in chondrocytes. We found that loss of Fgfr3 aggravated articular cartilage destruction mainly via chondrocyte hypertrophy and ECM degradation. Chondrocyte hypertrophy induces the expression of catabolic factors in articular cartilage that initiate and precipitate cartilage degeneration during OA development (27). In humans, gain-of-function mutations in FGFR3 result in skeletal dysplasia characterized by short stature due to inhibited proliferation and differentiation of growth plate chondrocytes (7). Interestingly,
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although patients with achondroplasia exhibit severe bowleg deformity and a higher incidence of obesity, they rarely develop OA (13,50). One possible reason for this is that activation of FGFR-3 signaling inhibits chondrocyte hypertrophy in articular cartilage in a manner similar to inhibition of terminal chondrocyte differentiation in growth plates. In our study, we found that FGFR-3 negatively regulated the expression of hypertrophy markers such as MMP-13 and type X collagen in articular chondrocytes. These results indicate that FGFR-3 delays OA development by suppressing articular chondrocyte hypertrophy in adults. The decrease in ECM in aging cartilage may also be due to reduced protein synthesis in chondrocytes (51). FGF-9 and FGF-18, two high-affinity ligands that specifically activate FGFR-3 in chondrocytes, have been reported to be involved in cartilage synthesis (41,52,53). We found that Col2a1 and aggrecan expression in articular chondrocytes was down-regulated in the absence of Fgfr3, suggesting that FGFR-3 also prevents articular cartilage from degeneration via an anabolic effect. However, the detailed mechanism of the anabolic effect of FGFR-3 signaling on chondrocytes remains unclear. We previously showed that IHH signaling acts downstream of FGFR-3 signaling in growth plate chondrocytes (33,54), and it was recently reported that the expression of IHH and Gli1, a downstream effector of IHH signaling, is up-regulated in the articular cartilage of OA patients, which is correlated with up-regulation of MMP13, COL10A1, and ADAMTS5 (31). Furthermore, transgenic mice with overactivation of IHH signaling have cartilage damage with increased chondrocyte hypertrophy resembling human OA (31). In contrast, Ihh deletion in chondrocytes delays the development of OA induced by DMM surgery (32). These findings demonstrate that IHH signaling contributes to OA development. In this study, expression of Ihh and Gli1 was increased in Fgfr3-deficient mouse cartilage explants. Furthermore, treatment with an IHH signaling inhibitor suppressed the increase in Mmp13, Col10a1, and Adamts5 levels in Fgfr3-deficient mouse cartilage explants. In addition, activation of IHH signaling has been reported to increase cartilage expression of RUNX-2, a critical transcription factor for chondrocyte hypertrophy and catabolism (31). It has previously been shown that the expression of RUNX2 is down-regulated in primary chondrocytes from patients with thanatophoric dysplasia type II resulting from a gain-of-function mutation in FGFR3 (55). In the present study, we found that Fgfr3 deficiency induced Runx2 expression in mouse articular chondrocytes, and the Runx2 expression was abrogated by the inhibition of IHH signaling. Thus, we
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propose that FGFR-3 suppresses chondrocyte hypertrophy and preserves ECM content in part via an IHH– RUNX-2–dependent mechanism. FGFR-1 and 3 are highly expressed in adult human articular chondrocytes (10,11). We previously found that Fgfr1 deletion protects adult cartilage from degeneration (9) and that a peptide or chemical antagonist of FGFR-1 can prevent the development of OA in mice (56), suggesting that FGFR-1 and 3 have opposite effects on articular cartilage maintenance; although the underlying mechanism is unclear, it can be speculated that these effects are mediated via differential activation of downstream signaling pathways (40). Interestingly, Wang et al (57) proposed that FGFR-1 and 3 may have similar downstream signaling pathways in cartilage development, since the mice they generated with chimera FGFR composed of activated extracellular and transmembrane domains of FGFR-3 and intracellular domain of FGFR-1 also exhibited retarded long bone development, similar to that of mice with an activation mutation in FGFR-3 (57). Thus, another possible explanation for the different roles of FGFR-1 and 3 in articular cartilage is the different patterns of their spatiotemporal expression. This question remains to be studied by using multiple approaches such as genetic modulation of FGFR spatiotemporal expression in different cell lineages of articular cartilage. In summary, by using both inducible chondrocytespecific activation and inactivation approaches, we found that Fgfr3 deficiency in chondrocytes exacerbates DMMinduced OA development, while gain-of-function of FGFR-3 delays OA progression. These findings strongly suggest that FGFR-3 plays a chondroprotective role in joints in the adult mouse, which will aid in the understanding of the mechanism of OA and facilitate the development of effective treatment strategies for this common disease. ACKNOWLEDGMENTS The authors thank Huabin Qi, Xiaofeng Wang, Min Jin, Qiaoyan Tan, and Xiaogang Li (Daping Hospital, Third Military Medical University, Chongqing, China) for technical support, and David Cushley (International Science Editing) for assistance with manuscript preparation. 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. Drs. L. Chen and Xie had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Tang, Su, L. Chen.
Acquisition of data. Tang, Su, Zhou, Xie, Huang, Wen, Z. Wang, Q. Wang, Xu, Du, H. Chen. Analysis and interpretation of data. Tang, Su, Zhou, Xie, L. Chen.
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