| Phosphorus plays an extremely important role in ensuring the eggshell quality of poultry,maintaining normal bone metabolism,and reducing osteoporosis at the end of egg production.As a bone-derived hormone,Fibroblast growth factor 23（FGF23）inhibits the reabsorption of phosphate in the kidneys of mammals,and promotes the excretion of phosphate.It is a key factor in regulating phosphorus metabolism.Therefore,studying how FGF23 regulates the phosphorus metabolism and skeletal development of poultry is of great significance for understanding the mechanism of FGF23 and the poultry production industry.In this thesis,we initially studied the factors that affecting FGF23 expression and the direct effect of overexpressing FGF23 on osteoblasts.We have initially identified the role by which FGF23regulates mineralization of chicken osteoblasts.Part 1:Factors affecting the expression of FGF23 in osteoblastsBone marrow mesenchymal cells（BMSCs）were cultured in vitro and induced to differentiate into osteoblasts.On the 8th day of induced differentiation,a large number of mineralized nodules were observed and the nodules were stained red by alizarin red S staining solution,indicating that BMSCs were induced to differentiate into osteoblasts.In order to explore the factors that affect the expression of FGF23,osteoblasts were treated withβ-Glycerol phosphate sodium（β-Gly,5mM,10mM,20mM）,Calcium chloride（CaCl2,5mM）,1,25（OH）2D3(10-9M,10-8M,10-7M),and parathyroid hormone(PTH,10-9M,10-8M,10-7M)for 48h.The results showed thatβ-Gly,CaCl2 and 10-9M 1,25（OH）2D3 significantly inhibited the expression of FGF23（P<0.001）,10-8M 1,25（OH）2D3 had no effect on the expression of FGF23,but 10-7M 1,25（OH）2D3 and PTH significantly up-regulated FGF23 expression（P<0.001）.The above results showed that the effects of 1,25（OH）2D3 and PTH on the expression of osteoblasts FGF23 were consistent with previous reports in mammals,but the effects of CaCl2 andβ-Gly on the expression of FGF23 in osteoblasts were different from mammals.Part 2:Effect ofβ-Gly,CaCl2 and 1,25（OH）2D3 on osteoblasts during differentiation stageWe usedβ-Gly（5mM）,CaCl2（5mM）and 1,25（OH）2D3(10-7M)to treat osteoblasts while inducing BMSCs to differentiate into osteoblasts,and then investigated the effects of calcium,phosphorus and vitamin D on the expression of FGF23 and mineralization in osteoblasts.Tested genes include:FGF23;Runx family transcription factor 2（Runx2）;ectonucleotide pyrophosphatase/phosphodiesterase 1（ENPP1）,ectonucleotide pyrophosphate/-phosphodiesterase 3（ENPP3）that controls the production of inorganic pyrophosphate（PPi）,ANK（progressive ankylosis）that controls the transport of PPi from intracellular to extracellular,non-specific alkaline phosphatase（TNAP）,osteopontin（OPN）,Phosphate regulates neutral endopeptidase（PHEX）.The results showed thatβ-Gly significantly increased the mRNA expression of ALP and ENPP3（P<0.05）,and significantly inhibited the mRNA expression of FGF23,Runx2,ENPP1,ANK,TNAP,OPN,and PHEX（P<0.05）;CaCl2 significantly increased the mRNA expression of FGF23,Runx2,ALP,ENPP1,ANK,OPN,and PHEX（P<0.05）,significantly inhibited ENPP3 and TNAP mRNA expression（P<0.05）;1,25（OH）2D3 significantly up-regulated FGF23,ALP,ENPP1,ANK,OPN,PHEX and VDR mRNA expression（P<0.05）,significantly inhibited TNAP mRNA expression（P<0.05）,1,25（OH）2D3 have no effects on Runx2 and ENPP3 mRNA expression.Alizarin red staining showed that 1,25（OH）2D3 treatment significantly reduced the number of mineralized nodules（P<0.05）.Protein expression test results showed that the P-ERK and P-ERK/ERK protein expression levels were up-regulated significantly by 1,25（OH）2D3（P<0.05）.The above results suggested that,for osteoblasts at the differentiation stage,β-Gly significantly inhibited the expression of FGF23 and other genes related to intracellular deposition of PPi;CaCl2 significantly promoted the expression of FGF23 and other genes related to intracellular deposition of PPi;1,25（OH）2D3 significantly up-regulated the expression of FGF23 and other genes related to intracellular deposition of PPi,and significantly inhibited osteoblasts mineralization.The ERK signaling pathway was involved in this regulation.Part 3:The role and mechanism of FGF23 on osteoblastsTo investigate the direct effect of FGF23 on differentiation and mineralization of osteoblasts,FGF23 adenovirus（Adv-FGF23,107 pfu/mL）was overexpressed at different growth stages of osteoblasts.We used FGF/VEGF receptor tyrosine kinase inhibitor（50nM）to block FGFR3（Fibroblast growth factor receptor 3）,and explored the mechanism by which FGF23 plays a regulatory role.The results hinted that the overexpression of FGF23 significantly reduced the number of osteoblast mineralized nodules（P<0.001）,the expression of OPN mRNA was significantly up-regulated（P<0.001）,the expression of TNAP mRNA was significantly down-regulated（P<0.001）.FGF23 significantly inhibited alkaline phosphatase（ALP）activity（P<0.001）.The protein expression levels of P-ERK and P-ERK/ERK were significantly up-regulated（P<0.001）.FGFR3 inhibitor treatment partially reversed the inhibitory effect of FGF23,specifically by reducing the expression of FGF23 and OPN mRNA（P<0.001）,restoring ALP activity to the control level（P<0.001）,and inhibiting ERK phosphate（P<0.001）.FGF23 had no reversal effect on TNAP mRNA expression.The above findings evidenced that FGF23 has a significant inhibitory effect on the mineralization of osteoblasts,and the ERK signaling pathway was involved in the regulation of osteoblasts mineralization by FGF23.In summary,FGF23 is an inhibitor of osteoblasts mineralization.Calcium,phosphorus,vitamin D and parathyroid hormone all regulate FGF23 expression in chicken osteoblasts in vitro.FGF23 acts on different growth stages of osteoblasts,reduces the differentiation potential of BMSCs by reducing the ALP enzyme activity through the FGFR3.FGF23inhibits the generation and absorption of phosphates,reduces the production of mineralized nodules,and thereby inhibiting osteoblasts mineralization.ERK signaling pathway was involved in the regulation of osteoblasts mineralization by FGF23. |
