Vitamin D/Vitamin D Receptor Regulates Bone Metabolism Through Modulating Mannose Receptor C Type 2 In Mouse

Objectives:Vitamin D plays an important role in maintaining skeletal development and bone homeostasis.It,on the one hand,indirectly regulates bone homeostasis through intestine and kidney,on the other hand,directly regulates bone metabolism through osteoblasts.Although vitamin D has been extensively studied,the direct effect of vitamin D on osteoblasts remains unclear.In this study,we performed tandem mass tag experiments on MC3T3-E1 cells treated with and without1,25?OH?₂D₃ to explore the 1,25?OH?₂D₃ action on murine osteoblasts and analyze the roles and mechanisms of 1,25?OH?₂D₃/VDR in bone metabolism.Methods:This research is divided into three parts.?1?Identification and analysis of proteins regulated by 1,25?OH?₂D₃ in mouse osteoblasts:The mouse osteoblast cell line MC3T3-E1 was treated with active vitamin D?1,25?OH?₂D₃?and ethanol,respectively.TMT experiments were carried out to identify differentially expressed proteins in these cells,and the differentially expressed proteins were analyzed by bioinformatics.Some differentially expressed proteins related to bone metabolism were screened for Western blot verification and analysis.?2?Spatiotemporal localization and expression of MRC2 in fetal mouse femur were studied,and further analysis of regulatory relationship between Vitamin D/VDR and MRC2 in osteoblasts:In the fetal mouse femur,the expression of MRC2 was localized by immunohistochemistry.In the model mouse lacking active vitamin D?Cyp27b1knockout mouse?the expression of MRC2 was analyzed.In the mouse osteoblasts cell line MC3T3-E1,1,25?OH?₂D₃ were added exogenously after the VDR was suppressed by siRNA or overexpressed by the adenovirus vector.Then the expression of MRC2in the osteoblasts was detected to analyze the relationship between 1,25?OH?₂D₃/VDR and MRC2.The binding activity of Vdr to Mrc2 promoter region was analyzed by double luciferase reporter gene assay.?3?The effect of 1,25?OH?₂D₃/VDR/MRC2 on collagen metabolism in mouse osteoblastsIn MC3T3-E1,Mrc2 siRNA interferes with the expression of MRC2 and analyses the changes of collagen metabolism in osteoblasts;PRM method quantitatively analyses the effect of 1,25?OH?₂D₃ on the expression of collagen protein in osteoblasts;Combining the results of bioinformatics and PRM analysis,we regulate the expression of MRC2 in mouse osteoblasts MC3T3-E1,explore collagen-related proteins downstream of MRC2,and explore the relationship between MRC2 and collagen metabolism in osteoblasts.The mechanism of action of collagen-related proteins.Results:The results of the first part1.For TMT,129 proteins were significantly down-regulated and 150 proteins were significantly up-regulated in 1,25?OH?₂D₃–treated MC3T3-E1 cells.2.The molecular function of these significantly regulated proteins was mainly associated with protein binding,catalytic activity and a molecular function regulator.The significantly regulated proteins were mainly involved in a single organism process,biological regulation and the regulation of biological processes.KEGG pathway analysis indicated that the significantly regulated proteins were mainly enriched in the PI3K–Akt signaling pathway,focal adhesion,and protein digestion and absorption pathways.PPI analysis showed that some proteins related to collagens with high degree of connectivity may constitute key points affecting metabolic or signal transduction pathways of the entire system.3.The expression of three proteins related to bone metabolism?MRC2,WWTR1 and RASSF2?all increased in MC3T3-E1 cells treated with 1,25?OH?₂D34.The expression levels of MRC2 in MC3T3-E1 cells treated with different concentrations of 1,25?OH?₂D₃ was increased in a dose-dependent manner.The results of the second part1.In embryonic mouse femur,MRC2 was mainly expressed in the mast cell area of growth plate,bone formation area and ossification area.2.The expression of MRC2 was found to be decreased in the femur of Cyp27b1KO mice compared with wild-type mice.3.1,25?OH?₂D₃ failed to up-regulate the expression of MRC2 when the expression of VDR was suppressed in MC3T3-E1 cells.4.With overexpression of VDR in MC3T3-E1 cells,the up-regulation effect of1,25?OH?₂D₃ on MRC2 was significantly enhenced.5.Vdr,the transcription factor,was not capable of activating the Mrc2promoter.The results of the third part1.Interference with the expression of MRC2 induced an increase in extracellular matrix collagen in MC3T3-E1 cells.2.In MC3T3-E1 cells,1,25?OH?₂D₃ could significantly down-regulate the expression of COL5A2 and COL12A1.3.in MC3T3-E1 cells,the expression of COL5A2 increased with inhibition of MRC2 and co-IP showed that MRC2 interacted with COL5A2.4.We verified the co-expression of MRC2 and MMP13 during mouse embryonic development by at the protein level by immunofluorescence.The expression of MMP13 decreased in osteoblasts after the inhibition of MRC2 in MC3T3-E1 cells.Co-IP showed that MRC2 interacted with MMP13.Conclusions:1.1,25?OH?₂D₃ could up-regulate the expression of MRC2,WWTR1 and RASSF2 in osteoblasts.The expression levels of MRC2 was increased in a dose-dependent manner.2.The expression of MRC2 was decreased in the femur of Cyp27b1 KO mice with deficiency of 1,25?OH?₂D₃.3.The up-regulation of MRC2 expression in osteoblasts by 1,25?OH?₂D₃depends on VDR,but it is not achieved through the activation of VDR transcription.4.1,25?OH?₂D₃/VDR directly regulates collagen uptake by MRC2.5.1,25?OH?₂D₃/VDR/MRC2 regulates the expression of COL5A2 to negatively regulate the formation of collagen fibers.6.1,25?OH?₂D₃/VDR/MRC2 regulates the expression of MMP13 to participate in the catabolism of bone matrix collagen.