Role Of FGFR1 Signaling In Bone Development And Remodeling In Mice

FGF receptor 1 has an important role in bone development and congenital diseases in human. Fgfr1 (P252R) mutation causes Pfeiffer syndrome. A rare mutation has been identified that causes osteoglophonic dysplasia (OD), a disease characterized by craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions. Loss of function mutations in fgfr1 is associated with one form of Kalmann syndrome (KS), a disease that does not directly affect skeletal development. Fgfr1 knockout leads to embryonic lethality shortly after gastrulation, necessitating a conditional knockout approaches to address fgfr1 function in later development. Conditional knockout technology makes it possible to investigate FGFR1 function in bone devemopment. Osteocalcin-Cre (OC-Cre) transgenic mice are suitable for studying fgfr1 function in differentiated osteoblast, which highly express Cre at 4 to 8 week after birth. Fgfr1 has recently been shown to be pivotal for early limb bud development and distal skeleton patterning. How FGFR1 regulates osteoblast and bone quality are not clear yet. FGFR1 is one of FGF23's receptor, which implies a potential role of FGFR1 on calcium and phosphate metabolism.ALK3, one of BMP type I receptor, is a negative regulator of bone mass. Bone mass was increased in Alk3OC-CKO mice. Crosstalk between BMP signaling and FGF signaling are complicated and controversial in bone development. Conditionally Inactivating BMPRIA and BMPRIB showed that BMP and FGF coordinately regulate chondrogenesis, BMP signaling inhibits FGF signaling. FGF promotes BMP signaling by inhibiting BMP antagonist noggin. Crosstalk between FGFR1 and ALK3 is not clear. The aim of this project is to study the role of FGFR1 in bone development and the crosstalk between FGFR1 and ALK3 signaling.To explore the role of FGFR1 in differentiated osteoblast in bone development after birth and its mechanism, OC-Cre transgenic mice and fgfr1 floxed mice were used. And conditional caALK3 transgenic mice were used for constitutively activated ALK3 in fgfr1OC-CKO mice. Two parts of this project were included: Part one: To investigation the role of FGFR1 in osteoblast in bone development and remodeling and its mechanism.Part two: To investigation the relationship between FGFR1 and ALK3 in differentiated osteoblast.Main experiments show below:Part one: Role of FGFR1 signaling in bone development and remodeling Experiment animal: fgfr1OC-CKO mice and fgfr1CKO miceMethods:In vivo assay1. Obtaining of loss of FGFR1 function in differentiated osteoblast mice (fgfr1OC-CKO mice) and general phenotype analysis Fgfr1OC-CKO mice were obtained according to what we reported. X-Ray photograph was taken for different ages. Whole skeleton prepared according to litterature.2. Analysis of fgfr1OC-CKO mice and control mice femurFemur of 3 and 8 month-old mice were scanned byμCT 80 (scanco). Condition: 70KVp, 113μA, resolution: 20μm. Integration time: 400msec.3. Histology analysisFemurs were collected from 3 month-old mice, 24 hrs fixation, and paraffin embedding for section. H&E staining and TRAP staining were taken. SEM was applied for bone and osteoblast morphology analysis.4. Osteoid, phosphate and bone formation rate analysisUndecalcified sections were used for Von Kossa staining. Calcein was used for bone formation rate detection. EDS was applied for Ca and P content and percentage analysis.5. Serum Ca and PO4 detectionSerum calcium and phosphate were analyzed by Beckman DXC800 auto biochemistry analysis machine and kit. 6. Mechanical testThree bending approaches were applied to analyze 3 and 8 month-old mice femur mechanical characteristics. Speed 6 mm/min, Maximal load 5 kN load cell.In vivo assay1. Primary calvarial osteoblast proliferation, mineralization, and apoptosis detectionPrimary calvarial osteoblasts were separated from neonatal mice. Osteoblast proliferation, mineralization, and apoptosis were detected.2. Osteoblast gene transcription analysis by real time PCRPrimary calvarial osteoblast was maintained in mineralization medium for two weeks, and total RNA was extracted. Osteoblast gene transcription of osteocalcin,osteopontin,cbfa1,noggin,bmp4,bmpr1a and fgfr2 were detected by real time PCR using SYBR.3. Osteoblast protein expression detectionTotal protein was extracted from primary calvarial osteoblast maintained in mineralization medium for two weeks. Phospho-p38, phospho-ERK, ERK, phospho-SAPK/JNK, p85, BMPRIA and internal control beta actin expression were detected by western blot.Part two: Conditionally constitutively activated ALK3 in differentiated osteoblast can partially rescue fgfr1OC-CKO mice bone abnormalityAnimal: fgfr1OC-CKO-caALK3 mice, fgfr1OC-CKO mice and fgfr1CKO mice (control)Methods: all the methods in this part were described in part one.Main results:1. Bone formation was increased after deleting FGFR1 in differentiated osteoblastLoss of FGFR1 signaling led to significantly increased BMD, cortical bone thickness BV/TV and trabecular bone at 8 month-old mice, though 3 month mice had no difference in cortical bone thickness compare with control mice, but had much more trabecular bone. HE staining results showed that osteocytes were impaired in FGFR1 conditional knockout mice. In vitro osteoblast mineralization test also showed that lacking of FGFR1 in differentiated osteoblast led to increased mineralization. In vivo double labeling results showed increase bone formation, which was consistent with in vitro data. 2. FGFR1 regulates bone quality partially through ALK3 signalingAlthough bone formation was significantly increased at 8 month-old mice, part of 3 month-old mice displayed spontaneous fracture. And the mechanical properties were decreased, in the middle of cortical bone, some osteoid bone was found by Von Kossa staining and counterstained with Van Gieson's staining. To our surprise, low mechanical properties of fgfr1OC-CKO mice could be partially rescued by constitutively activated ALK3 in differentiated osteoblast.3. FGFR1 signaling and BMP signaling can coregulate serum calcium and phosphateFgfr1OC-CKO mouse showed increase serum calcium and phosphate, especial the serum phosphate level. At 4 and 8 month-old mice, serum phosphate was increased 30.1% and 27.4% separately. Calcium and phosphate disorder of fgfr1OC-CKO mouse could be rescued by constitutively activated ALK3 in differentiated osteoblast.4. FGFR1 indirectly promotes osteoclast function, ALK3 active osteoclast function.Compared with control mice, fgfr1OC-CKO mice showed decreased osteoclast number and osteoclast size. Activation of ALK3 could turn over this situation.5. FGFR1 can regulate osteoblast proliferation and apoptosis through PI3KWestern blot results showed that p85 expression, the p-ERK, p-p38 and p-SAPK-JNK level were increased in fgfr1OC-CKO mice primary calvarial osteoblast. And the apoptosis of osteoblast lacking FGFR1 was decreased by TUNEl, the proliferation was in creased.6. FGFR1 can regulate BMPRIA and BMP4 expression in osteoblastGene expression and transcription detection results showed that BMP4 transcription and BMPRIA transcription and expression were down regulated in the osteoblast lacking of FGFR1.7. FGFR1 signaling regulate bone formation through MAPK signalingWestern blot results showed that phospho-p38, phospho-ERK and phospho-SAPK/JN were deceased, but the ERK level did not changed in fgfr1OC-CKO mice primary calvarial osteoblast. This suggested that FGFR1 can affect bone formation through MAPK signaling.Conclusions:1. Disruption of FGFR1 in differentiated osteoblast-specific led to mice tibia spontaneous fracture, decreased biomechanical properties, and could be rescued by constitutively activated ALK3 in differentiated osteoblast. 2. FGFR1 promotes apoptosis through PI3K and MAPK, and FGFR1 inhibits osteoblast proliferation and mineralization.3. FGFR1 inhibits serum phosphate and calcium metabolism by promoting ALK3 signaling.4. FGFR1 deficiency in osteoblast led to decreased osteoclast activity, and could be rescued by constitutively activated ALK3 in differentiated osteoblast.