The Role Of The Vitamin D Receptor In Bone Marrow Mesenchymal Stem Cells In Regulating Bone Mass

Objectives:1,25-dihydroxyvitamin D（1,25（OH）₂D₃）is the active form of vitamin D,which plays a wide range of physiological regulatory roles by activating the vitamin D receptors（VDRs）in many target organs throughout the body.When VDRs in intestinal mucosa and renal tubular epithelial cells are activated,the absorption and reabsorption of calcium in the intestinal and renal tubules are increased,which is beneficial to preventing bone loss.Therefore,VDRs in intestinal mucosa and renal tubular epithelial cells have a positive regulatory effect on bone mass and is beneficial to bone health.However,the knockout of VDR in mouse osteoblasts showed an increase in bone mass,suggesting that VDR in osteoblasts has a negative regulatory effect on bone mass and is detrimental to bone health.However,it is unclear whether VDR in bone marrow mesenchymal stem cells（BMSCs）,the precursor of osteoblasts,also have negative regulatory effects that are detrimental to bone health.Clarifying the role of VDR in BMSCs will help optimize therapeutic strategies for bone metabolic diseases such as osteoporosis and seek new therapeutic targets.This study aims to clarify the role of VDR in BMSCs and explore the underlying mechanism.Methods:BMSCs VDR conditional knockout mice（c KO）were successfully constructed by breeding with Cre/Lox P system.At 8 weeks,homozygous control mice and c KO mice were sacrificed by neck amputation.The growth,body length,general appearance and weight of these mice were observed.The femurs of the two types of mice were dissected and isolated,and the length and size of the femurs were observed.The heart,liver,spleen,lung,kidney,colon,skeletal muscle and bone tissues of these mice were collected,and RNA was extracted and quantitative PCR（q PCR）was used to examine the expression of VDR in various tissues and organs.BMSCs were isolated from these mice and cultured.The RNA and protein were extracted,and the expression levels of VDR at RNA and protein levels were examined.H&E staining was used to observe the histomorphology of heart,liver,spleen,lung,kidney,colon,fat and skeletal muscle.The femurs of Control and c KO mice were reconstructed using micro computed tomography（μCT）to analyze the bone microstructure of cancellous bone and cortical bone.Calcein double labeling test was used to examine the amount of bone formation in control and c KO mice at 7 days.The bone mineral deposition rate was calculated according to the calcein double labeling test and the bone mineral deposition rate.Alkaline phosphatase（ALP）staining and osteocalcin（OCN）staining were used to label femoral osteoblasts from control mice and c KO mice.TRAP staining was used to label the femoral osteoclasts of control mice and c KO mice.The morphology of femur,bone trabecula and bone marrow adipose cells were examined by H&E staining.The knee joints of control and c KO mice were stained with safranin O and fast green to examine the histology of knee cartilage and cartilage matrix.BMSCs of wild-type C57 mice were extracted by the bone marrow adherent method.Bone formation and lipid formation were induced,and alizarin red and oil red O staining were performed 14 days later.The primary BMSCs from control mice and c KO mice were isolated and cultured.The plates were seeded and cultured and osteogenic induction was carried out.q PCR was used to measure the m RNA levels of RANK,RANKL,andβ-catenin in BMSCs from control and c KO mice after 1,25（OH）₂D₃intervention.Results:There was no significant difference in growth and development,body length,general appearance,and body weight between the two types of mice.There was no significant difference in femur length and size.There was no significant difference in VDR expression in heart,liver,spleen,lung,kidney,colon and skeletal muscle between the two groups of mice.The expression of VDR in bone tissue of c KO mice was significantly lower than that of control mice.VDR expression in BMSCs of BMSCs conditioned knockout mice was significantly lower than that in BMSCs of control mice at both the m RNA and protein levels,suggesting that BMSCs VDR-c KO mice were successfully constructed.HE staining showed that there was no significant difference in the morphology of heart,liver,spleen,lung,kidney and colon.The morphology of adipocytes in white adipose tissue of c KO mice was fuller than that of control mice,suggesting that the differentiation of adipose tissue of c KO mice was stronger than that of control mice.The muscle fibers in skeletal muscle tissue of c KO mice were sparse compared with those of control mice,and the gaps between muscle bundles were enlarged,suggesting that the differentiation of skeletal muscle tissue in c KO mice was reduced.TheμCT analysis showed that the number,density and structure of trabecular bone in c KO mice were increased,and the bone microstructure was improved.The cortical bone thickness of c KO mice was thicker than that of control mice.BMD,bone volume percentage（BV/TV）and trabecular bone number（Tb.N）of c KO mice were significantly lower than those of control mice.The trabecular bone thickness（Tb.Th）of c KO mice was higher than that of control mice,but the difference was not statistically significant.The trabecular separation（Tb.Sp）in c KO mice was lower than that in control mice,but the difference was not statistically significant.The percentage of femoral cortical bone volume（BV/TV）and cortical bone thickness（Cort.Th）of c KO mice were higher than those of control mice.The total porosity[porosity（Po）,Po（tot）]of c KO mice was lower than that of control mice.Cortical bone volume BMD was not significantly different between the two groups.The calcein double labeling test showed that the distance between the double labeled fluorescence in the femur of c KO mice was significantly smaller than that of control mice.The mineral deposition rate of c KO mice was lower than that of control mice.These results suggest that c KO mice have reduced bone formation,and VDR promotes osteogenic differentiation.Alkaline phosphatase（ALP）staining showed that the number of positive staining cells（purple red）on the surface of trabecular bone in c KO mice was significantly lower than that in control mice,suggesting that the number of osteoblasts is reduced.OCN immunohistochemistry showed that the number of positive staining（brown）cells on the surface of bone trabecula in c KO mice was significantly lower than that in control mice.These results showed that BMSCs VDR conditional knockout mice（c KO）had fewer osteoblasts and weakened osteogenic differentiation,suggesting that VDR stimulates osteogenic differentiation.Tartrate-resistant acid phosphatase（TRAP）staining showed that the number of positive staining（purple red）cells on the surface of trabecular bone in c KO mice was significantly reduced,suggesting that osteoclast differentiation in c KO mice is weakened and VDR stimulates osteoclast differentiation.H&E staining of femur showed that the number of femoral bone trabeculae of c KO mice was increased compared to that of control mice,and no significant changes were observed in adipose cells.Red solid green staining of knee joint showed an increase in the number of red positive chondrocytes and the amount of cartilage matrix in c KO mice,and increased cartilage differentiation in c KO mice.These results suggest that VDR inhibits differentiation of BMSCs into chondrocytes,but has no effect on the adipose differentiation of BMSCs.Red positive calcium nodules were examined by alizarin red staining and the results showed that BMSCs extracted by bone marrow adherent method differentiated into osteoblasts.Red positive lipid droplets were examined by oil red O staining and the results showed that BMSCs extracted by bone marrow adherent method differentiated into adipocytes.These results validated the methods of BMSC extraction.After induction of osteogenic differentiation,1,25（OH）₂D₃stimulated RANKL expression and inhibited RANK expression in BMSCs,and these effects disappeared in BMSC isolated from c KO mice,suggesting that VDR activation stimulates RANKL expression and inhibits RANK expression in BMSCS.Conclusions:VDR in BMSCs promotes bone formation and bone resorption and reduce bone mass in mice.At the same time,VDR increases the level of RANKL expression in BMSCs and decrease the level of RANK expression in BMSCs.This regulation may participate in the mechanism by which VDR in BMSCs negatively regulates bone mass.