Exogenous PTH1-34 Can Rescue Achondroplasia And Delayed Fracture Healing Caused By Gain-of-function Mutation Of FGFR3

A dozen or more activated mutations in the coding sequence of the FGFR3 gene can cause a variety of human dwarfism with developmental disorders,such as achondroplasia (Ach) ,hypochondroplasia (HCH), thanatophoric dysplasia (TD), and some other related disorders.ACH is the most common form of dwarfism and a kind of autosomal dominant inheritable disease.The characteristic phenotypes of ACH include rhizomeric short limbs, macrocephaly, and lumbarlordosis.There is no effective therapy for ACH so far other than bone lengthening.Chen et al made a knock-in mouse model (FGFR3G369C) mimicing achondroplasia and found that this model had the bone phenotypes, such as small habius, short and round skull, structural abnormality of growth plate, disturbed proliferation and differentiation of growth plate chondrocytes and so on. Deng et al identified the role of FGFR3 in endochondral ossification by disrupting the murine FGFR3 gene.FGFR3 knock-out mice (FGFR3-/-) exhibited enhanced and prolonged endochondral bone growth accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. FGFR3 appeared to regulate endochondral ossification by an essentially negative mechanism .Chen et al reported that FGF-FGFR3 signaling targets STATs and IHH to inhibit chondrocyte proliferation , while FGF-FGFR3 singnaling and PTHrP/IHH singaling may inhibit chondrocyte differentiation independently. It has been found that both Ach-(FGFR3 G380R) and TDII-type(FGFR3 K650E) mutant FGFR3 induce apoptotic changes and marked decrease of PTHrP expression in ATDC5 cells, a mouse chondrogenic cell line.PTHrP treatment can block the apoptotic response induced by FGFR3. Bone culture study has suggested that PTH1-34 inhibited differentiation and apoptosis of chondrocytes and improved bone growth,which indicates that PTH1-34 may be used as a potential therapeutic agent for achondroplasia.Whether or not the systemic administration of PTH1-34 is a pharmacological therapy for ACH needs to be further studied.As a regulator of bone growth, FGFR3 is also involved in the regulation of fracture repair.Su et al reported that gain-of-function mutation of FGFR3 resulted in delayed fracture healing by inhibiting chondrocyte differentiation and bone resorption. Bone lengthening,a frequently performed surgery in patients with ACH, also involves fracture healing process. Thus ,searching for the biological measure to improve the bone healing in patients with ACH is very important.Recombined human PTH1-34(Teriparatide) is the FDA-approved drug to treat osteoporosis in humans by promoting bone formation. There have been a variety of studies on the role of PTH1-34 in fracture healing,which demonstrated that PTH1-34 preferentially enhanced chondrocyte recruitment the rate of chondrocyte maturation to stimulate endochondral ossification. Whether or not PTH1-34 is an effective biological therapeutic measure for the delayed fracture repair in ACH needs further study.In this study, we used mouse model mimicking human achondroplasia caused by gain-of-function mutation of FGFR3 (FGFR3G369C/+ mice) to explore the effect of PTH1-34 on the achondroplasia and delayed fracture healing resulting from ACH.MethodsPart I Study of the effects of exogenous PTH1-34 on the bone development retardation of FGFR3G369C/+ mice1. Animal model: New born wild-type(WT) and ACH mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.2. The body weight as well as length of naso-anal, tibia and femur were measured, whole skeleton staining and skull photography were performed. The cranial synchondrosis and foramen magnum of mice were observed.3. Histological sections of mice at different ages were stained with HE and safranine-fast green . Chondrocyte proliferation in the growth plate was investigated by BrdU labeling assay. 4. The lengths of hypertrophic chondrocyte zone were measured. The expression of some marker genes during chondrocyte differentiation including typeⅡcollagen, type X collagen and PTHrP were detected by real-time PCR.5. The bone culture system of metatarsal in vitro was established, metatarsal growth rates with or without PTH1-34 were determined and compared.6. Intermittent PTH1-34 treatment were performed in primary cultured chondrocytes. Cell proliferation was detected using an in vitro MTT colorimetric assay and cytometry. The expression levels of FGFR3 were detected by real-time PCR and western blotting.Part II Study of the effects of exogenous PTH1-34 on the delayed fracture healing of FGFR3G369C/+ mice1. Closed fracture of proximal tibia was created and stabilized with an intramedullary pin in 7-8-week-old mice. The mice were randomly divided into treatment group and control group. The mice in treatment group were given PTH1-34 80μg/(kg·d) subcutaneously until the end of the observation period while the control mice were given sterile water.2. Callus tissues were analyzed at 1-4 weeks post-fracture by radiography and histology.3. The concentration of serum calcium and phosphorus were measured.4. RNA was isolated from callus tissues, and the expression levels of bone formation-related genes were evaluated by real-time PCR.ResultsPart I Exogenous PTH can rescue the retarded bone development in ACH mice caused by carrying gain-of-function mutation of FGFR31. The naso-anal lengths of ACH mice treated by PTH1-34 at 4th week were significant longer than that in ACH control mice.The cranial synchondrosis time of ACH mice treated by PTH1-34 was delayed compared with that in ACH control mice.The size of foramen magnum had no significant difference.2. The chondrocyte proliferative index measured by BrdU labeling assay indicated that the chondrocyte proliferative index in ACH mice treated by PTH1-34 was significantly higher than that in ACH control mice. The cell culture study confirmed this result.3. On postnatal 7 d and 10 d, the hypertrophic zone of chondrocytes in growth plate of PTH1-34-treated ACH mice was wider than that in ACH control mice, and the secondary ossification center was observed advanced in PTH1-34 treated ACH mice..Real-time PCR measurement of both tissues and cells had comfirmed that.4. Analysis suggested that the tibial growth rate of ACH mice was significantly slower than that in littermate WT control. PTH1-34-treatment can improve the growth suppression in ACH mice.5. The expression of FGFR3 was decreased by PTH1-34 treatment.Part II Exogenous PTH1-34 can rescue the delayed fracture healing caused by gain-of-function mutation of FGFR3 in mice1. At 7 days post-fracture, cartilaginous callus areas were increased in PTH1-34-treated ACH mice compared with those of control mice. In contrast, at 14 days post-fracture, the remnant cartilaginous callus areas were smaller in PTH1-34-treated mice than those in control mice. There were remnant cartilaginous callus in ACH control mice while little remnant cartilaginous callus was observed at 21 days after fracture. The remnant fibrous bone areas in marrow cavity were also reduced in PTH1-34-treated mice compared to control mice at 28 days after fracture.2. In the early stages of fracture (7 days), compared with the control group, the PCNA and COL10A1 mRNAs expression were obviously increased in PTH1-34 treatment group. In the middle stages of fracture(14 days), compared with the control group, the OC mRNA expression level were significantly increased in PTH1-34 treatment groupConclusions:1. The gross phenotypes of ACH mice treated with PTH1-34 including body weight and body length were increased compared with that in ACH control mice.2. The proliferative and differentiation activity of chondrocytes in growth plates of ACH mice were inhanced by intermittent PTH1-34 treatment.3. Results of PTH1-34 treatment on metatarsals suggested that PTH1-34 could promote the bone elongation of ACH mice.4. The rescuing effect of PTH1-34 on bone retardation of ACH mice may be related to down regulation of FGFR3 expression.5. Exogenous PTH1-34 promoted bone fracture healing in terms of increasing callus areas, endochondral ossification.