The Roles And Mechanisms Of FGFR3 In Both Cartilage Development And Homeostasis

Fibroblast growth factor receptor(FGFR)3, as a transmembrane receptor, serves essential roles in the early stages of cartilage development. Patients with activating FGFR3 mutations exhibit skeletal dysplasias characterized by short stature including achondroplasia(ACH, OMIM 100800), thanatophoric dysplasia I/ II(TD I, OMIM 187600 and TD II, OMIM 187601), hypochondroplasia(OMIM 146000), and severe ACH with developmental delay with acanthosis nigricans(SADDAN, OMIM 187600). In contrast, a loss-of-function mutation in FGFR3 in humans causes camptodactyly, tall stature, and hearing loss(CATSHL) syndrome(OMIM 610474). Thus, FGFR3 is considered to negatively regulate endochondral bone growth during cartilage development, including the proliferation and differentiation of chondrocytes. However, the role and mechanism of FGFR3 in other skeletal diseases remain largely unknown.Benign cartilaginous tumors, including osteochondroma and enchondroma, are the most frequently primary tumors affect skeletal system. Cartilaginous tumors are frequently developed around growth plates during skeleton development, indicating that they may be resulted from disordered endochondrial bone growth. Multiple cartilaginous tumors can be found in several hereditary diseases, such as hereditary multiple exostoses syndrome(HME, also named hereditary multiple osteochondromas, OMIM 133700), enchondromatosis(ollier diseases and Maffucci syndrome, OMIM 166000) and metachondromatosis(MC, OMIM 156250), which are caused by mutations in the genes, including EXT1/2, PTPN11, PTHR1, IDH1/2. Interestingly, most genes involved in the development of benign cartilaginous tumors are associated with FGFR3 signaling pathway. Furthermore, it is reported that osteochondromas have been observed in the bones of members of a CATSHL family. These evidences strongly imply that FGFR3 may contribute to the development of cartilaginous tumors. However, the direct role and mechanism of FGFR3 in the development of cartilaginous tumors remain unclear.The terminal differentiation of chondrocytes, also called chondrocyte hypertrophy, play a critical role in the initiate and progression of osteoarthritis(OA) during adult stage. Recently, inhibition of articular chondrocyte hypertrophy is considered as an effective way to delay or prevent OA development. In our previous studies, FGFR3 mainly expressed in the proliferating and hypertrophic zones of growth plate, which inhibits the early and terminal differentiation of growth plate chondrocytes. In addition, FGFR3 expression was detected in articular chondrocytes, which is gradually downregulated during OA progression. Thus, FGFR3 not only regulates the early cartilage development, but also involves in the homeostasis maintenance of articular cartilage during adult stage. Given to the role of FGFR3 in the inhibition of chondrocyte hypertrophy, we speculate that FGFR3 may protect articular cartilage from degeneration. Therefore, we use genetic mouse model to study the effect of FGFR3 on the homeostasis maintenance of temporomandibular joint(TMJ) cartilage during adult stage.Post-traumatic osteoarthritis(PTOA) is a common disease in joints. Due to the chondroprotective role of FGFR3 in articular cartilage during OA development, we speculate that FGFR3 activation can be a potential therapy to delay cartilage degeneration during PTOA development. It is previously reported that FGF9 can specifically activate FGFR3 signaling pathway in chondrocytes. Thus, we performed the intra-articular injection of exogenous FGF9 in mice with surgery-induced PTOA to investigate the role of FGF9 in PTOA development. We found that exogenous FGF9 significantly ameliorates the loss of chondrocytes and extracellular matrix in articular cartilage of mice with surgery-induced PTOA.Methods:Part I Study of the role of FGFR3 in the development of cartilaginous tumors1. Inducible chondrocyte-specific knockout of Fgfr3 in mice during adolescence: Tamoxifen(TM, 1mg/10 g body weight) was intraperitoneally injected twice weekly in mice at age of 4 weeks for 8 weeks.2. The body length and the length of long bones were respectively measured. The morphologic analysis of the skeleton in Fgfr3-deficient mice were performed by X-ray and micro-computed tomography(Micro-CT). The histological analysis of the skeleton in Fgfr3-deficient mice were performed by hematoxylin/eosin(HE) and Safranin O/Fast green stain. Immunostaining analysis was performed to detect the expression of PCNA, Ki67, collagen X, MMP13, IHH, ERK and acetylate tubulin in growth plates and chondromas. TUNEL assay was also performed to investigate the apoptosis of growth plate chondrocytes by an In Situ Cell Death Detection kit.3. Primary growth plate chondrocytes were isolated from knee joints of Fgfr3 cKO and Cre-negative mice at age of 3 days. The qRT-PCR analysis was performed to detect the mRNA expression of Col10, Mmp13, Adamts5, Spp1, Ihh and Pthrp in primary chondrocytes. Western blot analysis was also performed to detect the protein expression of phospho-ERK in primary chondrocytes;4. Inhibition of Indian Hedgehog(IHH) signaling in vivo: An IHH signaling inhibitor(GDC0449, SMOi) was used in our study. Fgfr3 cKO and Cre-negative mice are randomly divided into 4 groups: Cre-negative mice treated with Vehicle, Cre-negative mice treated with SMOi, Fgfr3 cKO mice treated with Vehicle and Fgfr3 cKO mice treated with SMOi. GDC0449 was reconstituted in 50%(w/v) 2-hydroxy-propyl-β-cyclodexyrin in water. Mice were i.p. injected twice weekly with TM in mice at age of 4 weeks and daily i.p. with GDC0449(1mg/g body weight) or 50%(w/v) 2-hydroxy-propyl-β-cyclodexyrin(Vehicle).5. Dynamic observation of survival in mice with SMOi treatment. The length of body, tibia and femur in mice with SMOi treatment were respectively measured. The morphologic analysis of the skeleton in mice with SMOi treatment were performed by X-ray and micro-computed tomography(Micro-CT). The histological analysis of the skeleton in mice with SMOi treatment were performed by hematoxylin/eosin(HE) and Safranin O/Fast green stain.Part II Study of the role of FGFR3 in osteoarthritis development at temporomandibular joint of mice during adult stage1. Inducible chondrocyte-specific knockout of Fgfr3 in mice during adult stage: Tamoxifen(TM, 1mg/10 g body weight) was intraperitoneally injected 5 days in mice at age of 8 weeks.2. The morphologic analyses of skull and temporomandibular joint(TMJ) in Fgfr3 cKO mice are performed by X-ray and micro-computed tomography(Micro-CT). The histological analysis of TMJ cartilage in Fgfr3 cKO mice were performed by HE and Safranin O and Fast green stain. Immunohistochemistry staining were performed to detect the expression of FGFR3, collagen II, PCNA, collagen X, MMP13, ADAMTS5, Aggrecan, Lubricin, RUNX2 and IHH in TMJ cartilage and disc. TUNEL analysis was also performed to detect the apoptosis of TMJ chondrocytes.3. Primary TMJ chondrocytes were isolated from Cre-negative and Fgfr3 cKO mice at age of 4-5 days. The qRT-PCR analysis was performed to detect the mRNA expression of Col10a1,Mmp13,Adamts5,Gli1 and Runx2 in primary TMJ chondrocytes.4. IHH signaling Inhibition in vivo: Mice were i.p. injected twice weekly with TM in mice at age of 2 months and daily i.p. with GDC0449(1mg/g body weight) or 50%(w/v) 2-hydroxy-propyl-β-cyclodexyrin(Vehicle).5. The histological analysis of TMJ cartilage in mice with SMOi treatment were performed by HE and Fast green and Safranin O staining.Part III Intra-articular injection of exogenous FGF9 ameliorates DMM-induced PTOA in mice1. Immunohistochemistry staining were performed to detect the expression of FGF9 in the cartilage from mice with DMM surgery.2. Human full-thickness articular cartilage: Human articular cartilage tissues randomly divide into 4 groups: Control group, FGF9 treatment group, IL-1β treatment group and IL-1β+FGF9 treatment group. The histological analysis of human cartilage tissues is performed by Safranin O staining. Immunohistochemistry staining were performed to detect the expression of collagen II,collagen X and MMP13 in these articular cartilage tissues.3. Primary human articular chondrocytes: Human articular chondrocytes randomly divide into 4 groups: Control chondrocytes group, FGF9 treated chondrocytes group, IL-1β treated chondrocytes group and IL-1β+FGF9 treated chondrocytes group. Western blot was performed to detect the expression of Aggrecan and MMP13 in these human articular chondrocytes.4. Surgery-induced PTOA mouse model: Destabilization of the medial meniscus(DMM) surgery was performed, which induces the destabilization of knee joints in mice. The C3 H background mice were used in our study. DMM surgery was performed in right side of joints in the littermates of male mice, and Sham operation was performed in the left side of joints in these mice.5. Intra-articular injection of exogenous FGF9 in mice: Joints of mice randomly divide into 4 groups: Sham operation with vehicle treatment group, Sham operation with FGF9 treatment group, DMM surgery with vehicle treatment group and DMM surgery with FGF9 treatment group. At 2 weeks after DMM surgery, intra-articular injection of 5 μl FGF9(1 μg in 5 μl saline) or 5 μl saline was performed in mice.6. The morphologic analysis of subchondral bone in mice with DMM surgery or Sham operation after saline and FGF9 treatment were performed by Micro-CT scan. The histological analysis of articular cartilage in these mice were performed by Safranin O and Fast green stain, and an osteoarthritis research Society International(OARSI) score system was used to quantify the OA severity in mice. Immunohistochemistry staining were performed to detect the expression of collagen II, collagen X, MMP13 and Cleaved caspase-3 in articular cartilage and the expression of Collagen II, PCNA and SOX9 at the sites of osteophyte formation.Results:Part I Chondrocyte-specific knockout of Fgfr3 in mice directly induces the formation of metachondromatosis-like lesions1. Chondrocyte-specific knockout of Fgfr3 significantly increases the body length and the length of tibia and femur in mice during adolescence. Multiple chondroma-like lesions were observed in Fgfr3-deficient mice. The histological analysis found that Fgfr3 deficiency leads to the disorder of growth plate chondrocytes, the formation of enchondroma-like lesions in bone marrow and osteochondroma-like lesions in cortical bone.2. Chondrocyte-specific knockout of Fgfr3 leads to the polarity loss, increased proliferative activity, promoted differentiation and decreased apoptosis of chondrocytes in growth plates.3. Chondrocyte-specific knockout of Fgfr3 significantly downregulated p-ERK expression and upregulated IHH/PTHrP expression in chondrocytes.4. Inhibition of IHH signaling significantly ameliorates the phenotypes of multiple chondroma-like lesions in Fgfr3 cKO mice.Part II FGFR3 signaling involves in the homeostasis maintenance of TMJ articular cartilage during adult stage1. No significant change was observed in the gross morphology of skull and TMJ of mice after chondrocyte-specific knockout of Fgfr3 during adult stage. However, histological analysis revealed that chondrocyte-specific knockout of Fgfr3 during adult stage causes TMJOA-like phenotypes, including articular chondrocyte loss, cartilage extracellular matrix loss and fissure formation in superficial zone of TMJ articular cartilage.2. Chondrocyte-specific knockout of Fgfr3 during adult stage significantly increased the expression of collagen X, MMP13 and ADAMTS5 in TMJ articular cartilage, while decreased the expression of aggrecan and lubricin. In addition, TUNEL-positive cells were significantly increased at superficial zone of TMJ articular cartilage in mutant mice. The bone mineral density of subchondral bone was significantly increased in mutant mice. We found that there is no significant change of chondrocyte proliferative activity in mutant mice.3. Chondrocyte-specific knockout of Fgfr3 during adult stage significantly increased IHH signaling and the expression of RUNX2 in TMJ articular chondrocytes.4. IHH signaling inhibitor significantly ameliorates TMJOA-like phenotypes result from chondrocyte-specific knockout of Fgfr3 in mice during adult stage.Part III Intra-articular injection of exogenous FGF9 significantly ameliorates the phenotypes of DMM-induced PTOA1. The expression of FGF9 was significantly downregulated in articular cartilage from mice with DMM surgery.2. Intra-articular injection of exogenous FGF9 significantly ameliorates the OA phenotypes, including articular extracellular matrix loss, the increased expression of Collagen X and MMP13. Moreover, exogenous FGF9 significantly downregulated the matrix loss and the increased expression of collagen X, MMP13 and ADAMTS5 in human articular cartilage tissues with IL-1β.3. Intra-articular injection of exogenous FGF9 significantly aggravates the osteophyte formation in mice with DMM surgery.4. The expression of collagen II, SOX9 and PCNA was significantly increased at sites of osteophyte formation in mice with DMM surgery after FGF9 treatment.Conclusion:1. Chondrocyte-specific knockout of Fgfr3 during adolescent stage in mice directly induces the formation of multiple chondroma-like lesions.2. Chondrocyte-specific knockout of Fgfr3 during adult stage in mice causes TMJOA-like phenotypes, at least partly, via the upregulation of IHH/RUNX2 signaling pathway.3. Intra-articular injection of exogenous FGF9 protects articular chondrocytes from degeneration during PTOA development, but aggravates osteophyte formation.