Vitamin D/Vitamin D Receptor Regulates Skeletal Development Through Modulating Calbindin D28K

Objectives:Body calcium and bone homeostasis are closely linked together.The integrity of the skeletal structure depends on an adequate supply of calcium in the bloodstream,but also indirectly from intestinal calcium absorption and kidney calcium reabsorption.Calcium,on the other hand,can also be released from bone to maintain blood calcium levels at negative calcium balance.The calcium-binding protein 28K?CaBP 28K?plays an important role in the regulation of calcium transport and its relationship with the vitamin D/vitamin D receptor?VDR?in bone has not been elucidated.This study was designed to investigate the role and mechanism of vitamin D/VDR and CaBP 28K in bone development.Methods:This research is divided into three parts.?1?Serological analysis of the correlation between 25?OH?D₃ and CaBP 28K and their correlation with fetal bone development:The content of 25-hydroxyvitamin D3[25?OH?D₃]in blood serum was measured by ELISA kit.The growth and development indexes of neonates were collected:body weight?WT?,body length?BL?,head circumference?HC?,chest circumference?CC?,length of femur?FL?,biparietal diameter?BPD?.The correlation between 25?OH?D₃and CaBP 28K in serum of pregnant women and neonates was analyzed.The correlation between 25?OH?D₃ and CaBP 28K in serum and skeletal development indexes of neonates was also analyzed.?2?Spatiotemporal localization and expression of CaBP 28K in fetal mice femur were studied,and further analysis of osteoblastic regulatory relationship between Vitamin D/VDR and CaBP28K in osteoblasts:In the newborn mouse femur,the expression of CaBP 28K was localized by immunohistochemistry and immunofluorescence,and the tendency of CaBP 28K expression was analyzed with the change of gestational age.In the mouse osteoblastic cell line MC3T3-E1 and the human osteoblast cell line hFOB1.19,different concentrations of 1,25?OH?₂D₃ were added exogenously.After the VDR was overexpressed at the gene level by the adenovirus vector,the change of CaBP 28K in osteoblasts was analyzed,and the binding activity of VDR to CaBP 28K promoter region was analyzed by luciferase.Meanwhile,the change trend of CaBP 28K in the femur of1?hydroxylase knockout mice was analyzed.?3?Intervene the expression of CaBP28K and VDR in osteoblasts,screen the expression changes of the downstream indicators,and determine the content of the downstream indicators screened in the same batch of serum samples to analyze its correlation with CaBP28K:In the mouse osteoblastic cell line MC3T3-E1,the expressions of CaBP28K and VDR were regulated,the changes of osteoblasts behavior were analyzed,and the changes of downstream osteoblast differentiation,mineralization and proliferation were analyzed.Results:The results of the first part1.The content of 25?OH?D₃ in maternal serum and neonatal umbilical serum was positively correlated?N=58,R=0.836,P=0.000?2.The content of CaBP28K in maternal serum and neonatal umbilical serum was positively correlated N=56,R=0.278,P=0.038;3.There was a significant negative correlation between the content of 25?OH?D3and CaBP28K in maternal serum,N=58,R=-0.308,P=0.019;4.The content of CaBP28K in maternal serum was negatively correlated with the content of 25?OH?D3 in neonatal umbilical blood serum?N=58,R=-0.365,P=0.005?5.The content of CaBP28K in maternal serum was significantly and positively correlated with neonatal body length,N=58,R=0.287,P=0.029;6.Neonatal cord blood serum CaBP28K content and neonatal body length,head circumference,chest circumference were significantly positive correlation.N=69?R=0.242,0.386,0.273;P=0.045,0.001,0.023?;The results of the second part1.In embryonic mouse femur,CaBP28K was mainly expressed in the growth plate mast cell area and the original osteogenic area;2.With the increase of fetal mouse gestational age?E14-E16-E18?,CaBP28K showed a gradually decreasing trend in the femur of embryonic mice;3.The expression of CaBP28K were down-regulated after the cells were stimulated with 1,25?OH?₂D₃ in MC3T3-E1 cells and hFOB1.19 cells;4.The expression of CaBP28K were down-regulated after the cells were transfected with the adenovirus over-expressing VDR in MC3T3-E1 cells and hFOB1.19cells;5.In mouse osteoblasts MC3T3-E1 and human osteoblast hFOB1.19,when transfected with VDR siRNA,the expression of VDR was decreased,while CaBP28K increased;6.Compared with wild type mice,the thickness of cortical bone in 1?hydroxylase knockout mice was significantly reduced;7.In the femur of 1?hydroxylase knockout mice femur,VDR expression decreased,CaBP28K expression increased;8.Dual luciferase reporter gene results showed that VDR has a binding site in the promoter region of CaBP28K.The results of the third part1.The overexpression of CaBP28K in mouse osteoblast MC3T3-E1 can inhibit its proliferation and apoptosis and promote its differentiation;2.The overexpression of CaBP28K in mouse osteoblast MC3T3-E1 resulted in the down-regulation of MMP13 expression and the up-regulation of DMP1 expression.Blockade of p38-MAPK signaling pathway blocked the modulation of MMP13 by CaBP28K.3.Protein interaction of CaBP28K and MMP13 exist in MC3T3-E1 cells;4.In mouse MC3T3-E1 cells,blockade of p38-MAPK signaling pathway could block CaBP28K's regulation of MMP13;5.There was a significant negative correlation between CaBP28K and MMP13 in maternal and neonatal cord blood serum,N=73,R=-0.251,P=0.0326.There was a significant positive correlation between the content of MMP13 and OPG and CTX in neonatal cord blood serum?N=85,R=0.22,0.26;P=0.04,0.01?.7.In maternal serum,CaBP28K was negatively correlated with OPN and CTX?N=58??R=-0.249,-0.264;P=0.025,0.045?;Conclusions:1.In the process of neonatal bone development,CaBP28K is positively correlatedwith the body length,head circumference,and chest circumference of newborns.CaBP28K is an important contributor to fetal bone development and is closely related to 25?OH?D₃;2.CaBP28K influences the growth and development of the embryonic mouse bone by regulating the proliferation,apoptosis and differentiation of the osteoblasts and the original bone formation region of the embryonic mouse bone growth plate;3.1,25?OH?₂D₃/VDR has a negative relationshipn with CaBP28K in bone and osteoblasts.CaBP28K maybe a middle bridge of the negative function of1,25?OH?₂D₃/VDR on the skeleton;4.1,25?OH?₂D₃/VDR regulates the expression of MMP13 and DMP1 through CaBP28K and influences skeletal development.The p38-MAPK pathway is involved in the regulation of MMP13.1,25?OH?₂D₃/VDR/CaBP28K/MMP13 is involved in the regulation of synthesis and catabolism during bone development.