Study On The Role Of FGF2 In Bone Homeostasis In Mice

Bone is a dynamic organ that is constantly remodeled,and bone homeostasis is maintained by a combination of osteocytes,osteoblasts,and osteoclasts.Osteoblasts are responsible for the synthesis,secretion and mineralization of bone matrix and eventually differentiate into osteocytes.Osteoclasts are responsible for the breakdown and resorption of bone.Disruption of bone homeostasis causes a range of skeletal disorders.For instance,when bone resorption exceeds bone formation,causing reduction in bone mass and the onset of osteoporosis.And osteosclerosis will develop when osteogenesis exceeds osteoclastogenesis.Therefore,maintaining the functional stability of osteoblasts and osteoclasts is an effective measure for preventing and treatment skeletal diseases.Although fibroblast growth factor 2（FGF2）regulates bone homeostasis,its specific mechanism remains to be further investigated.In this study,we tested the effect of FGF2 on mineralization by culturing bone marrow stromal cells（BMSCs）in vitro.Moreover,we supplemented SSR128129E（SSR）and AZD4547（AZD）to BMCs as well as administered SSR to mice by gavage to block FGF/FGFRs signaling and PD0325901（PD）to block ERK signaling to test their effect on osteoporosis.SSR and AZD are respectively allosteric and tyrosine kinase inhibitors of FGFs receptors（FGFRs）.PD,a blocker of ERK signal pathway.Besides,FGF2 knockout mice were used to verify the effect of FGF2 on bone homeostasis.The following were the main results:（1）The result of Alizarin red staining showed that exogenous addition of recombinant FGF2 inhibited the mineralization of BMSCs.AKP and TRAP enzyme activity assay indicated that the exogenous addition of FGF2 group had no significant difference in AKP enzyme activity compared with the control,but the TRAP enzyme activity was higher than that in control.（2）Western blot results showed that exogenous addition of recombinant FGF2 activated the phosphorylation of ERK and CREB and promoted the protein expression of osteoclastogenesis related genes,while the presence of PD inhibited the increased expression.The result of Alizarin red staining showed that PD antagonized the FGF2 inhibition of BMSC mineralization,the data of chromatin immunoprecipitation indicated that p-CREB binding to the promoter region of Rankl.（3）Exogenous addition of SSR was performed on BMSCs,but its reversal effect on FGF2 inhibition of mineralization was limited.However,exogenous addition of AZD to BMSCs almost completely blocked the inhibitory effect of FGF2 on mineralization.（4）RT-q PCR and Western blot analysis demonstrated that the m RNA and protein expression of osteoclastogenesis related genes were downregulated and the mineralization capacity was greater in BMSCs from FGF2 knockout mice compared with wild-type.The m RNA expression of osteoclastogenesis related genes and the phosphorylation level of ERK were downregulated in FGF2 knockout mice compared with wild-type.（5）Results from SSR administration test indicated that the BMD,BMC and the stout degree of femur and tibia were lower in the experimental mice than the control.Taken together,we determined that FGF2 negatively regulated bone homeostasis by enhancing osteoclastogenesis through in vitro and in vivo experiments.FGF2 activated ERK-CREB signaling,which in turn activated osteoclastogenesis that mediated by RANKL.AZD showed a strong reversal on the FGF2 effect in cellular assays,while the antagonism of SSR on FGF2’s action was weak.Our results provide new targets for the prevention and treatment of osteoporosis and ideas for the development of related drugs.