The Role And Mechanism Of Wnt And TGF-? Signaling Pathway In The Regulation Of 1,25?OH?₂D₃ On Bone Homeostasis

Osteoporosis is the most common metabolic bone disease that causes a heavy public health burden on the aging society.Because 1,25?OH?₂D₃plays an indirect role in maintaining the balance of blood calcium and phosphorus required for mineralization through the intestines,kidneys,and thyroids.Therefore,inadequate dietary intake of vitamin D has been considered an important risk factor for osteoporosis.But in recent years,some studies have shown that prophylactic vitamin D supplements not only fail to prevent osteoporosis,they even lower bone mineral density and increase the risk of fractures.Also,as the active ingredient of vitamin D,the direct effect and mechanism of1,25?OH?₂D₃on bone homeostasis are sill not clear.Therefore,we established animal models with different levels of vitamin D to investigate whether vitamin D deficiency can lead to osteoporosis.In vitro,1,25?OH?₂D₃was used to intervene in pre-osteoblasts and osteoclast precursors,to evaluate their regulatory effects on bone homeostasis and related mechanisms.In this study,we creatively constructed vitamin D-deficient mice and model mice that rescued this deficiency.The results showed that without causing changes in calcium and phosphorus levels,a reduction in vitamin D would not induce osteoporosis.In addition,calcein-fluorescent labeling and TRAP staining showed that the decreasing of vitamin D increased the mineral apposition rate and slowed bone tissue absorption.This result may provide some clues to explain that the high-dose supplementation of vitamin D found in clinical practice reduced BMD and increased the risk of fracture.In order to explore the regulatory effect of 1,25?OH?₂D₃on bone homeostasis,we performed MC3T3-E1,bone marrow mesenchymal stem cells?BMSCs?,and bone marrow macrophages?BMMs?with 1,25?OH?₂D₃,ALP staining and alizarin red staining tests revealed that early and late osteogenic differentiation of MC3T3-E1 and BMSCs were significantly inhibited after treated with 1,25?OH?₂D₃.At the same time,Alamar Blue,immunofluorescence staining,TRAP staining and Western Blot found that1,25?OH?₂D₃induced BMMs differentiate to osteoclasts.We have also established a co-culture system of BMSCs and BMMs,to further confirmed that 1,25?OH?₂D₃inhibits osteogenesis and promotes osteoclastogenesis in bone homeostasis.In order to further verify whether changes in vitamin D levels of the bodies would alter the response of cells to 1,25?OH?₂D₃,we extracted cells from model mice.Through a series of experiments in vitro,it was found that 1,25?OH?₂D₃played a role in suppressing osteogenesis and inducing the osteoclast mature.To address the molecular mechanism of the regulation of 1,25?OH?₂D₃on bone homeostasis,we analyzed the genes with variable expression after the treatment with1,25?OH?₂D₃by RNA sequencing.Among most up-regulated genes after treatment with1,25?OH?₂D₃are ones that are involved in osteoclast differentiation pathway and down-regulated genes are involved in the Wnt and TGF-?signaling pathways.Validated by a series of molecular biological methods,we conceived that 1,25?OH?₂D₃can inhibit the Wnt signaling pathway?LRP5-?-catenin-RUNX2?and TGF-?signaling pathway?BMP2-RUNX2-SP7?.At the same time,activated VDR induced osteoclast formation by activating the VDR of BMSCs and stimulating the secretion of RANKL.To summarize,the direct effect of 1,25?OH?₂D₃on bone homeostasis is to activate bone resorption and inhibit bone formation.