The Effect And Mechanism Of Regulating Bone Hematopoiesis By Osteocyte Conditional Knockout FGFR1

Background:Bone is a supportive organ for mechanics, and it can also influence metabolization, endocrine and hematopoiesis. Bone has abundant calcium, phosphorus and other inorganic substance, participating in salt metabolization. Bone can also secrete several factors to regulate energy metabolization, balance of glucose and phosphorus.Osteocytes are the largest number of cells in bone, and play very important roles in bone homeostasis. Osteocytes can perceive the mechanical signal, regulate mineralization, and sustain homeostasis around them. Osteocytes regulate differentiation and functions of osteoblasts and osteoclasts through their dendrite connection. Furthermore they can also regulate mineral metabolization by endocrine function. FGFR1 is an important member of FGFRs family, and play critical roles in bone development. FGFR1 mutation results in several hereditary diseases of bone. FGFR1 is widely expressed in periosteum, mesenchymal cells and osteoblasts at different stages. FGFR1 suppresses proliferation of mesenchymal cells and promote differentiation of osteoblasts in early phase of bone development, suppressing mineralization in late phase. Osteocyte is the terminal state of osteoblast, and could be regulated by FGFR1. Some studies indicated that FGFR1 participated in the regulation to osteocyte.Wnt/β-catenin is a key signal pathway to regulate functions of osteocytes. It can not only change bone mass, regulate endocrine function of osteocytes, but also control conduction of mechanical signal, sustain activity of cells. There are interaction and crosstalk between Wnt/β-catenin signal and FGF signal. Our previous studies also found that FGFR2 and FGFR3 can regulate the activity of Wnt/β-catenin signal. Therefore, we speculate that FGFR1 may also regulate osteocytes through Wnt/β-catenin pathway.Objective:To clarify the function of FGFR1 in regulating osteocytes. To study the mechanism that FGFRs regulating bone metabolize and body hematopoiesis from a new view, and provide new idea to explore FGF/FGFR signals for regulating hematopoiesis.Methods:1. Fetal mice were stained to find if there were changes in mutant group in 15.5d.2. Measuring height and weight to compare development.3. Bone mass and cortical thickness of femurs were scanned by MicroCT.4. Serum glucose, calcium and phosphorus were measured.5.The number of osteoblasts and osteoclasts were assessed by morphometry using decalcified and undecalcified bone sections of tibia.6. Double labeling was used to assess the bone formation.7. Isolation and culture of primary osteocytes.8. RNA extraction of femoral shaft and primary osteocytes to detect the gene expression.9. Scanning electron microscope and transmission electron microscope were used to observe osteocytes of 6 months old mice.10. TUNEL assay was used to study the apoptosis osteocytes.Results:1.There was no difference in bone development between the two groups.2.Bone mass was increased significantly in mutant mice.3.The serum levels of glucose, calcium and phosphorus have no difference between the two groups.4.Bone formation rate was enhanced remarkablely, and the average number of osteoblasts and osteoclasts was changed obviously.5.SEM and TEM analysis showed that osteocytes of mutant mice were changed to oval and had typical changes of apoptosis.6.In mutant osteocytes, the expression of FGF23,SOST,DMP1, β- Catenin, LEF-1,DVL and OPG increased, but AXIN2 and RANKL reduced. There was different result in the expression of DMP1 between femoral shaft and primary osteocytes.7.The number of empty lacunas was much more in mutant mice than wide mice.8.TUNEL asssy showed that the bone lacunas had more positive signal in mutant mice.9.The expression of caspase3 and caspase8 were increased significantly in mutant mice.Conclusions:1. FGFR1 deletion in osteocytes did not affect the development of fetal mice.2. Deletion of FGFR1 in osteocytes resulted in increased bone mass and acceleration of bone formation in adult mice.3. Osteocytes conditional deficient in FGFR1 could promote differentiation of osteoblasts and suppress differentiation and function of osteoclasts.4. FGFR1 deletion in osteocytes led to reduction of SOST expression, and then resulted in up-regulation of WNT signal.5. FGFR1 deletion in osteocytes promoted OPG expression and inhibited RANKL expression, which is also a reason for increased bone mass in mutant mice.6. FGFR1 deletion in osteocytes caused apoptosis of osteocytes.