The Role And Mechanism Of FGF23/FGFRs In Regulating Osteoblast Differentiation

Background:Fibroblast growth factor(FGF)belongs to the Heparin binding growth factor family(HBGM),and members of its family have many roles in cell and metabolic homeostasis.In the process of evolution,FGF can develop multiple members by differentiating the differences in functions and structures.Under the action of heparan sulfate proteoglycans(HSPGs),FGF binds to the extracellular regions IgII and IgIII of the fibroblast growth factor receptor FGFR,and plays a role in embryonic development and adult tissue remodeling to maintain homeostasis.The key role is to promote cell proliferation and population growth in an autocrine or paracrine manner.In addition,there are three kinds of FGF with endocrine effects,namely FGF15/19,FGF21,FGF23,among which FGF23,mainly derived from bone tissue,secreted by osteoblasts and bone cells,mainly regulates the metabolism of phosphorus in the body.The role.The binding of endocrine FGF to HSPGs is decreased,but FGF4223 is involved in Klotho.Compared with FGFRs alone,FGF23 has a higher affinity for FGF23,which can better activate downstream signaling pathways.Its biological activity.It plays an important role in the pathological process of chronic kidney disease(Chronic kidney disease,CKD-MBD,Mineral and bone disease).FGF23 and Klotho form Klotho/FGF23 axis,which together maintain the body.Mineral metabolism homeostasis.Phosphorus,as one of the main substances in the body,exists in the form of phosphate in the body,and together with calcium participates in various activities such as bone formation,acid-base balance,and material energy metabolism.As an important factor regulating phosphorus in the body,such as the FGF23-KLOTHO system is inhibited,it can increase phosphorus reabsorption,leading to hyperphosphatemia.On the contrary,after elevated blood phosphorus levels,FGF23 can be secreted into the blood by bone tissue,increase urinary phosphorus excretion,inhibit the synthesis of vitamin D,and negatively regulate blood phosphorus.Current research has long revealed that FGF23 plays an important role in regulating bone formation and maintaining bone homeostasis.However,the effects and molecular mechanisms of FGF23 on postnatal bone development and remodeling are not fully understood,and FGF23 has not been reported on how to affect osteogenic differentiation.We know that both FGFR1 and FGFR3 may be receptors for FGF23,but how FGF23 binds to FGFRs to play an osteogenesis role has become the focus of our research.Part one:Expression and role of FGFR1-3 in osteogenic differentiation;Part two:The role of fibroblast growth factor FGF23 in the regulation of glycolysis in osteogenic differentiation.Main experiments show below:Part one:the expression of FGFRs signaling pathway in mouse bone development and reconstruction and its mechanismExperimental method:1.Isolation and culture of primary osteoblasts and osteogenic inductionCranial osteoblasts were isolated from newborn mice born within 3 days,and osteogenic differentiation was detected by Alkaline phosphatase protein(ALP)staining/Alizarin red staining for 5,10,and 15 days.qRT-The expression of osteogenic differentiation-related genes COL 1?,RUNX2,and OPN was detected by PCR.2.Expression of FGFR1-3 during osteoblast differentiation;The total RNA of osteoblasts was extracted,and then the expression of FGFR1-3 was detected by qRT-PCR,and the expression of FGFR1-3 in osteogenic induction was also detected.3.Detection of osteogenic differentiation after blocking FGFRs signal;In the process of osteogenic induction of osteoblasts,the FGFR signal blocker BGJ398 was added,and the osteogenic differentiation ability of primary osteoblasts and osteoblasts MC3T3-E1 was detected by ALP/Alizarin red staining.qRT-PCR was used to detect the expression of COL 1?,RUNX2 and OPN in osteogenic differentiation genes.The expression of COL 1? and RUNX2 in osteogenic differentiation proteins was detected by Western blot.Part two: FGF23 regulates the glycolysis of osteoblasts.Experimental method:1.The effect of FGF23 on osteogenic differentiation ability;The first part of the successfully isolated primary osteoblasts and the osteoblast cell line MC3T3-E1 were treated with FGF23.After 5 days/15 days of osteogenic induction,ALP/Alizarin red staining was used to detect osteogenic differentiation,and osteogenesis was performed without FGF23.The induction group was used as the CONTROL group.2.Osteoblasts detect glycolytic glycol related indicators in the presence of FGF23;The first part of the successfully isolated primary osteoblasts and the osteoblast cell line MC3T3-E1 were treated with FGF23,and the supernatant was extracted 24 hours after 48 hours of treatment to detect glucose consumption,lactic acid production,and glycolysis levels.After 48 hours,Total cellular RNA was extracted,and the expression of glycolytic-related genes was detected by qRT-PCR,and the untreated group was used as the CONTROL group.Main experimental results:1.Detection of osteogenic differentiation in isolated primary osteoblasts;In primary osteoblasts,morphological observation revealed that osteoblasts were in good condition.ALP staining revealed a deep blue-violet color,alizarin red staining showed an increase in red calcium nodules,and qRT-PCR was used to detect COL 1? and RUNX2 during osteogenic differentiation.The genes related to osteogenic differentiation such as OPN indicated that the primary osteogenic separation was successful,and the parallel cultured osteoblast cell line MC3T3-E1 was repeated with the above method,and a consistent trend was found.2.Detection of the expression of FGFRs during osteogenic differentiation;The FGFs/FGFRs signal is an important signaling pathway regulating the development and homeostasis of the body.The expression of FGFR1-3 was detected by qRT-PCR.It was found that FGFR1-3 increased with time,and the osteoblast cell line MC3T3-E1 was cultured in parallel.Repeat the steps in the above method to find a consistent trend.3.Blocking the expression of osteogenic related genes after FGFR signalingIn the osteogenic induction process of osteoblasts,after adding FGFR signal blocker BGJ398,ALP staining showed that blue-violet light became shallow,alizarin red staining showed increased red calcium nodules,and qRT-PCR found COL 1? during osteogenic differentiation.The expression of RUNX2,OPN and other osteogenic differentiation-related genes was reduced.Western blot was used to detect the expression of COL 1? and RUNX2 during osteogenic differentiation,and the parallel cultured osteoblast cell line MC3T3-E1 was used to repeat the above steps.the trend of.4.Enhanced osteogenic differentiation after treatment with FGF23In the process of osteogenic induction of osteoblasts,after adding FGF23,ALP staining revealed that the blue-violet color became darker and the red calcium nodules increased,and the parallel cultured osteoblast cell line MC3T3-E1 was repeated by the above method,and the trend was found to be consistent.5.After treatment with FGF23,use the kit to detect the related indicators of glycolysisAfter adding FGF23 treatment,glucose consumption increased,lactic acid production increased,and glycolysis level increased compared with the untreated group.After 48 hours of treatment,the glycolysis-related genes PFKFB2,PFKFB3,PFKFB4,ENO2,ENO3 were found by qRT-PCR.The expression of LDHA was increased to varying degrees.Repeated steps were repeated with the above-mentioned osteoblast cell line MC3T3-E1,and a consistent trend was observed.Conclusion:1.Successful isolation of primary osteoblasts;2.The expression of FGFR1-3 increased gradually during osteoblast differentiation.The osteogenic differentiation of osteoblasts was attenuated after inhibition of FGFR signaling,suggesting that FGFR signaling promotes osteogenic differentiation.3,FGF23 promotes osteogenic differentiation of osteoblasts,which is related to FGF23 promoting osteoglycolysis of osteoblasts.