The Role And Mechanism Of FGFR3 In Articular Cartilage Homeostasis

Osteoarthritis is the most common type of arthritis and may be the primary cause of disability in elderly people.By now,there are few effective biological treatments to prevent OA.Understanding the molecular mechanisms of OA will facilitate the development of novel therapies for OA.However,the underlying mechanisms of OA are still inadequately understood.In deed,many pathways and molecules essential for the cartilage or joint development also participate in OA pathogenesis,provide novel insight into osteoarthritis pathology with potential for further progression into preclinical studies.Fibroblast growth factor(FGF)signaling pathway has been identified to play essential roles in the regulation of skeleton development.FGFs exert their effects by binding to FGF receptors(FGFRs).There are unique spatio-temporal expression patterns of FGFRs during development and adult stage in the skeleton.For example,expression of FGFR3 is observed in chondrocytes located in the central of the mesenchymal condensation during early development,and then in pre-hypertrophic zones of and proliferating of growth plates and articular chondrocytes.Mutations in FGFR3 have been found to be important for a variety of human genetic skeletal dysplasias.Gain-of-function mutations in FGFR3 may result in hypochondroplasia,achondroplasia and thanatophoric dysplasia,but loss-of-function mutation of FGFR3 leads to tall stature,camptodactyly,scoliosis and hearing loss(CATSHL)syndrome in humans.These phenotypes found in human patients were also confirmed in mice with respective various genetic modifications of Fgfr3.These facts suggest that FGFR3 may acts as negative regulator of endochondral bone development.The important roles of FGFR3 in skeletal development and human genetic skeletal dysplasias indicates that FGFR3 may play as a essential role in the maintenance of articular cartilage,which is supported by several other studies.FGFR3 is expressed in articular chondrocytes during adult stage.Previous study showed that FGFR3 expression was down-regulated in articular chondrocytes from patients with OA.Valverde,et al.showed that conventional Fgfr3 knockout(Fgfr3-/-)mice had early spontaneous arthritis.In addition,although severe bowleg deformity and higher incidence of obesity(well-known risk factors for OA)are exhibited in ACH patients,OA is hardly developed.All these results indicate that FGFR3 is a key molecule preventing articular cartilage from degeneration.Although articular cartilage is the major damaged tissue of OA,all tissues composing of joint as a whole tissue,such as synovium and subchondral bone,are involved in the progression of OA.Fgfr3-/-mice and ACH patients have mutant FGFR3 gene in all cells or/and tissues,and exhibit abnormal skeleton development and dysregulated bone quality,etc.All these features make it is difficult to exclude the potential involvement of the changes in tissues other than articular cartilage and the role of skeleton maldevelopment in the pathology of OA exhibited by Fgfr3-/-mice and ACH patients.More evidence need to demonstrate the direct role of FGFR3 in articular cartilage maintenance and its underlying mechanisms are needed.Methods:Part I:1.FGFR3 was specifically deleted or activated in articular chondrocytes during adult stage by using Fgfr3fl/fl or Fgfr3K644E/neo mice,respectively,in a tamoxifen-inducible manner.2.A destabilization of the medial meniscus(DMM)surgery was performed in these mice.Aging mice mimicking human achondroplasia(Fgfr3G369C/+)were also used to study the role of FGFR3 in age-associated spontaneous OA.3.Knee joint cartilage of these mice were histologically evaluated and scored by OARSI recommended system.4.Expressions of key genes associated with articular cartilage maintenance were assessed quantitatively in hip cartilage explants.5.The effects of IHH signaling inhibition on Fgfr3-deficient explants were also analyzed.Part II:1.The expression of FGF9 in articular cartilage with OA is detected by immunohistochemistry.2.The effects of intra-articular exogenous FGF9 injection on post-traumatic OA induced by the destabilization of the medial meniscus(DMM)surgery are evaluated.3.Cartilage changes and osteophyte formation in knee joints are investigated by histological analysis.Changes in subchondral bone are evaluated by microcomputed tomography.The effect of exogenous FGF9 on an interleukin-1β(IL-1β)-induced ex vivo OA model of human articular cartilage tissues is also evaluated.Results:Part I: FGFR3 Inhibits Osteoarthritis Progression in the Knee Joints of Adult Mice1.Conditional Fgfr3 deficiency aggravated DMM-induced cartilage degeneration.2.The expressions of MMP13 and collagen X were significantly up-regulated,while the expression of collagen II were significantly down-regulated in articular cartilage of Fgfr3-deficient mice.3.In contrast,conditional activating FGFR3 attenuated DMM-induced cartilage degeneration.Moreover,activating FGFR3(Fgfr3G369C/+)attenuated age-associated cartilage degeneration.4.IHH signaling and RUNX2 levels in articular chondrocytes were up-regulated after deletion of Fgfr3,while inhibition of IHH signaling down-regulated the increased expressions of Runx2,Mmp13,Adamts5 and Col10a1 in Fgfr3-deficient cartilage explants.Part II: FGF9 attenuates cartilage degradation and aggravates osteophyte formation in post-traumatic osteoarthritis1.FGF9 expression was down-regulated in articular chondrocytes of OA but ectopically induced at sites of osteophyte formation.2.Intra-articular injection of exogenous FGF9 attenuated articular cartilage degradation in mice after DMM surgery.3.Exogenous FGF9 suppressed collagen X and MMP13 expressions in OA cartilage,while promoted collagen II expression.4.Similar results were observed in IL-1β-induced ex vivo OA model.5.Intra-articular injection of FGF9 had no significant effect on the subchondral bone of knee joints after DMM surgery,but aggravated osteophyte formation.6.The expressions of SOX9 and Collagen II,and cell proliferation were up-regulated at sites of initial osteophyte formation in mice with exogenous FGF9 treatment.Conclusions:1.FGFR3 delays OA progress in knee joints,at least partly,via its down-regulation of IHH signaling in articular chondrocytes of adult mice.2.Intra-articular injection of exogenous FGF9 delays articular cartilage degradation in post-traumatic OA,while aggravates osteophyte formation.