The Roles Of Endogenous FGF23in Differentiation Of Osteoblasts Enhanced By Parathyroid Hormone In Vitro

Objective:Promoting osteogenesis was the pharmacological basis for parathyroid hormone (PTH) in treatment of osteoporosis, but the underlying mechanisms and influence factors were far from clarified. Osteoblasts expressed fibroblast growth factor(FGF)23, which was recently supposed as a negative regulator in osteoblast differentiation and matrix mineralization. PTH regulated the expression of FGF23in osteoblasts, and whether the expresion of FGF23influences the effect of PTH in osteoblast differentiation was not clear. To clarfy this issue in details, the RNA interference (RNAi) strategy was used to silence the expression of FGF23in rat calvarial derived osteoblasts and effects of rhPTH1-34were investigated in osteoblasts with or without FGF23knock down.Methods:1. The osteoblasts were isolated by enzyme digestion from the calvaria of neonatal SD rats and cultured in MEM medium containing10%charcoal stripped fetal bovine serum (CSFBS), and its osteoblastic character was identified by alkaline phosphatase (ALP) staining and alizarin red staining (ARS) for cell and mineralized nodule respectively in vitro.2. To determine the role of PTH in differentiation of osteoblast, the primary rat calvarial osteoblasts were treated with rhPTH1-34for3days in vitro and the changes of proliferation and alkaline phosphates (ALP) activity were measured by MTT and PNPP methods respectively. Meanwhile the expressions of ALP and OCN were determined in osteoblasts by real-time RT-PCR after rhPTH1-34treatment.3. To study the effect of PTH on the expression of FGF23, the primary rat calvarial osteoblasts were treated with10-9mol/L rhPTH1-34. The expression of FGF23were determined by real-time RT-PCR (for mRNA levels) and western blot analysis (for protein levels) respectively.4. To determine the effect of endogenous FGF23in PTH actions, the FGF23was transiently silenced by the shRNA method in osteoblasts and the expression levels of FGF23, ALP and OCN were determined in transcriptional levels after rhPTH1-34treatment. Results:1. Slightly stimulating effect of rhPTH1-34was showed on osteoblasts differentiation in vitro. The total ALP acitivities were increased while the cell ALP activities (calculated by OD405/OD570) were slightly but no significantly changed. Meanwhile the mRNA levels of ALP and OCN increased35%(P<0.05) and16%(P>0.05) respectively by10"9mol/L rhPTH1-34in2hours treatment.2. The FGF23expression was up-regulated in osteoblasts by10-9mol/L rhPTH1-34in transcriptional level.3. The up-regulation of FGF23by rhPTH1-34was blocked by the transfection of shRNA of FGF23. Further, the marked stimulating effects of rhPTH1-34on ALP and OCN expression (about1.8and5.8folds respectively) were showed in the FGF23silenced osteoblasts.Conclusions:1. Slightly stimulating effect of rhPTH1-34was showed on osteoblasts differentiation in vitro..2. The FGF23expression was up-regulated in osteoblasts by rhPTH1-34in transcriptional level.3. The endogenous FGF23might involve in osteogenesis regulated by PTH. The up-regulation of FGF23expression by PTH could suppress its stimulating effect on osteoblasts differentiation, and the underlying mechanisms remain to be clarified.