The Role And Mechanism Of FGFR3in Skeleton Development And Homeostasis

Mutations in the coding sequence of the FGFR3gene can cause autosomal dominanthuman skeletal disorders, such as achondroplasia(Ach),the most common form of dwarfism,thanatophoric dysplasia(TD)[1], and so on. While no effective clinical therapy is available forTD patients, current treatments for ACH patients are mainly growth hormone treatment anddistraction osteogenesis, which have limited effect with many complications.FGFR3was found to negatively regulate endochondral ossification. Chen et al reportedthat FGF-FGFR3signaling targets STAT1/p21and Ihh to inhibit chondrocyte proliferation,while FGF-FGFR3signaling and PTHrP/IHH signaling may inhibit chondrocyte differentiationindependently. Yamanaka found that both Ach-(FGFR3G380R) and TDII-type (FGFR3K650E) mutant FGFR3induce apoptosis and significant decrease of PTHrP expression inchondrogenic cell line. PTHrP treatment can blunt the apoptotic response induced by FGFR3.Ueda suggested that PTH1-34improve growth cultured bone from ACH mice, whichindicates that PTH1-34may be used as a potential therapeutic agent for achondroplasia.However, the actual in vivo effect of PTH signaling on the skeletal development of micemimicking human ACH/TD and its action mechanism are still not fully clarified. The purposeof this study was to elucidate whether the in vivo systemic administration of PTH can rescuethe underdeveloped skeleton of ACH and TDII mice.Recent studies suggest that the degeneration of endplates play key role in thepathogenesis of intervertebral disk (IVD) degeneration, but the molecular pathways are notfully elucidated..Although FGFR3signaling plays a critical role in regulating bonedevelopment and maintaining bone homeostasis, its roles in maintenance of IVD tissueremain poorly understood. In this project, we study the role of FGFR3in IVD homeostasisutilizing mouse models with genetically modified FGFR3. Brachydactyly means short fingers and toes relative to other parts of the body. Fivetypes (BDA–E) have been described by clinical situations. Isolated BDE has been attributedto mutations in HOXD13and PTHLH; however, more isolated BDE genes need to beidentified. Here, we studied a family with autosomal-dominant BDE, found a mutation of Xand did preliminary work on its function in chondrogenesis.Methods:Part I Study of the effects of PTH1-34on the bone development retardation ofACH and TDII1. Administration of PTH to mice For Fgfr3+/G369Cmice (ACH mice) and theirwild-type littermates, PTH was administered subcutaneously at the dose of100μg/kg bodyweight per day for4weeks after birth. PTH was also administered subcutaneously at the doseof100μg/kg body weight in pregnant Fgfr3+/K644Eneomice crossed with EIIa-Cre mice atE13.5. Untreated animals were injected with the same volume of vehicle alone as control.2. The survived thanatophoric dysplasia type II (TDII) mice with PTH treatment wererecorded. The body weight, as well as length of naso-anal,humerus,femur and were measured,whole skeleton staining photography were performed. The pictures of skull were scratched byX-Ray and micro-computed tomography (μCT).3. Histological sections of mice at different ages were stained with H.E. and the bonestructure and volume of the femoral distal metaphysis were scanned with μCT.4. The bone culture system of metatarsal in vitro was established, metatarsal growthrates with or without PTH1-34treatment were determined and compared.5. Intermittent PTH1-34treatment was performed in primary limb bud mesenchymalcells with micromass culture, and the cartilage nodules were counted then stained with Alcianblue.6. The plasmid carrying WT FGFR3and MT FGFR3with activating mutation weretransfected to ATDC5and HEK293T. Then PTH1-34was added, the expression tyrosinekinase activity of FGFR3were detected by Western Blotting and Co-immune precipitate.Part II The roles of FGFR3in maintenance of intervertebral disc (IVD)1. We induciblly inactivated FGFR3in collagen2-expressing cells including innerannulus fibrosus cells and endplate chondrocytes by injecting tamoxifen into mice with both floxed FGFR3alleles and Col2a1-CreERT2allele (hereafter referred to as Fgfr3Col2ER).Fgfr3Col2ERmice and controls (Fgfr3flox/flox) were administered with tamoxifen at2-month(1mg/10g body weight,×5days, i.p. injection), and were evaluated during development stageand then examined at3and4months old.2. Spine tissue specimens were analyzed by X-Ray, μCT and histological analysis.3. Expression levels of cartilage and bone formation-related genes in IVD tissue wereevaluated by real-time PCR.Part III The role of Mutant X in chondrogenesis1. The plasmid carrying WT and MT X transfected to ATDC5. Cell proliferation wasdetected using an in vitro MTT colorimetric assay and cytometry. The secretion of GAG wasdetected by Alcian Blue staining. the expression levels of cartilage differentiation-relatedgenes were evaluated by real-time PCR.2. The reporter genes of pthlh with different lengths were constructed. Reporter assaywas carried to detect that the transcriptional activities of pthlh.Results:Part I PTH1-34rescues the retarded bone development of ACH and TDII mice1. PTH1-34rescued the lethal phenotype of mice mimicking human thanatophoricdysplasia type II.TDII mice treated with PTH have small stature, dome-shaped skull, markedabnormal hypertrophic zone and less trabecular bone.2. PTH1-34increased the length of tibiae and femurs and alleviates the disorganizedgrowth plates, delayed secondary ossification, dome-shaped skull and osteopenia in ACHmice.3. PTH-treatment can improve the growth suppression of ACH mice in bone cultureand mainly increase the elongation of cartilage.4. The expression and kinase activity of FGFR3was decreased and the expression ofPTHrP was increase by PTH treatment.Part II The roles of FGFR3in the maintenance of intervertebral disc (IVD)1. The endplates of mice with gain-of-function mutation of FGFR3exhibit delayedmineralization. 2. Mice with conventional knockout and chondrocyte-specific knock out of FGFR3showed skeletal defects including kyphosis, scoliosis, crooked tails and curvature andovergrowth of long bones and vertebrae. These two knock out mice included extensiveosteophytes formation in vertebral body at adult age.3. Histologically, Fgfr3Col2ER mice developed early-onset of aging related cartilageloss and had bony tissue in the endplates at3-and4-month old. The histology of bone-liketissues resembled those found in ectopically formed bone, containing bone marrow,osteoclasts and mineralized osteoblasts. Immunohistochemical analysis demonstrated anotable reduction in Collagen2level and a significant increase in MMP13) and Col10expression in the IVDs of Fgfr3Col2ER mice. Gene expression analysis of IVDsdemonstrated that inactivation of FGFR3led to enhanced Col10, Mmp13and Adamts5expression, and depressed Col2and Aggrecan expression.Part III Mutant X inhibits the chondrogenesis and transcriptional activities ofpthlh1. Mutant X inhibited the proliferation, matrix secretion in chondrocytes and theexpressions of Col2, Col10, Aggrecan, PTHrP and FGFR3.2. Mutant X led to depressed transcriptional activities of pthlh through its AP1combining site possibly.Conclusions:1. Intermittent PTH (1-34) injection rescues the retarded skeletal development andpostnatal lethality of mice mimicking human achondroplasia and thanatophoric dysplasia.2. FGFR3may have a protective effect during the degeneration of endplates/IVD.3. Mutant X may lead to chondrodysplasia through its inhibition on the proliferationand differentiation of chondrocytes by depressing the transcriptional activities of pthlh andup-regulating FGFR3expression.