The Expression And Local Mechanisms Of FGF23 Regulation In Rat Osteoblasts

Objective:To investigate the expression patterns of fibroblast growth factor 23 (FGF23)in osteoblast in a culture model and its local mechanisms, especially the roles of the local production of 1α,25 dihydroxyvitamin D and/or the cell communication in FGF23 up-regulation in differentiated osteoblasts.Methods:1.The primary rat calvarial osteoblasts were cultured in vitro and identified with ALP and alizarin red staining. FGF23 mRNA and protein levels were detected by RT-PCR and Western blot analysis. The confluent osteoblasts were treated with 5mmol/L CaCl2, 5mmol/Lβ-glycerophosphate,10"9 mol/L rhPTH(1-34) and 10-8 mol/L 1,25(OH)2D3 respectively for 3 days, and same volume of medium was added as the control.FGF23 mRNA levels were determined after the treatment.2. The cultures were monitored under microscopy and the cells were harvested in half-confluence, confluence, osteoid deposition and osteoid mineralization stages respectively. FGF23 mRNA and protern levels were determined by Real-time PCR and Western blot analysis. The ALP, OCN and OPN mRNA levels were also evaluated.3.CYP27B1 and CYP24A mRNA levels in cells of half-confluence and confluence were determined by real-time PCR. To investigate the impact of local 1,25 dihydroxyvitamin D, FGF23 expressions were further determined in osteoblasts in serum-free medium with or without 25-hydroxycholecalciferol (25-(OH)D, the substrate of 1α-hydroxylase) and the inhibitory effects of Ketoconazole (KET, the inhibitor of 1α-hydroxylase) were also evaluated.4. FGF23 expressions were examined in osteoblasts in serum-free medium with or without the carbenoxolone(CBX), which is an inhibitor of gap junction Cx43.Results:1.FGF23 mRNA and protein are positively expressed in cultured primary rat calvarial osteoblasts as determined by RT-PCR and WB analysis. The marked stimulating effect (about 16 times)(p <0.001)on FGF23 expression was showed by exogenous10-8 mol/L 1,25(OH)2D3 treatment while no significantly differences in FGF23 mRNA levels by CaCl2,β-glycerophosphate, rhPTH(1-34) treatments when compared with the control.2. Compared with the proliferating cells in half-confluence, the FGF23 expressions in the confluent differentiated cells were up regulated obviously by 7.5 fold (p<0.001)in mRNA level and 126% in protein level as determined by real-time RT-PCR and Western blot analysis. After that, FGF23 mRNA was back to the level in half-confluent cells after transiently up regulated at cell confluent stage, while the protein level accumulated and peaked during the osteoid deposition phase. FGF23 protein level were up to 207%(p<0.05) in half-confluent cells compared with the proliferating cells, and which suggested that expression levels of FGF23 in osteoblasts was cell development stage dependent. In regular culture condition, the mRNA levels of CYP27B1 (the gene code for 1α-hydroxylase enzyme which catalyse the conversion of 1,25(OH)2D3 from its inactive form,25(OH)D) and CYP24A (the gene code for 24-hydroxylase, a sensitive indicator of calcitriol action) were significantly increased by 2 fold and 34 fold respectively in the differentiated osteoblasts compared with the proliferating cells, which suggested intensive function of endogenous 1,25(OH)2D.3.When cultured in serum-free medium, the FGF23 mRNA levels in osteoblasts markedly increased in proliferating cells(16 fold) (p<0.001)and in differentiated cells (28 fold) (p<0.001) by the physiological dose of 25-(OH)D3 treatment. Meanwhile the CYP27B1 was slightly but significantly up regulated(p<0.05)and CYP24A was dramatically stimulated by 1700 fold (p<0.001)and 800 fold(p<0.001)respectively in transcriptional levels. The stimulation of 25-(OH)D3 on FGF23 expression was blocked by an inhibitor of the hydroxylase, 10nmol/L ketoconazole treatment.4. The FGF23 mRNA level was reduced by 38%(p<0.05) in confluent differentiated cells by 100μmol/L carbenoxolone treatment, while slightly but no significant reduction (p>0.05) showed in half-confluent cells.Conclusions:1.FGF23 expression was powerfully stimulated by exogenous 1,25(OH)2D in cultured rat osteoblast in vitro.2.The FGF23 expression in osteoblast is developmental stage-related and up-regulated in confluent differentiated osteoblasts. 3.The up-regulation of FGF23 expression in confluent differentiated cells might be related to autocrine/paracrine action of the osteoblast-derived 1α,25 dihydroxyvitamin D and the cell communication.