The Impact And Mechanism Of PRELP On The Osteoblastic Differentiation In MC3T3-E1 Cells

Mineralization is a complex process orchestrated by the balanced action of promoters and inhibitors of calcification including many mineralization-relevant proteins. The proline/arginine-rich end leucine-rich repeat(LRR) protein(PRELP) is a 58-k D glycosaminoglycan(GAG)-and collagen-binding anchor protein which belongs to a subfamily of LRR proteins. PRELP is unique in having a basic amino-terminal domain rich in arginine and proline residues, its N-terminal region binds heparin and heparan sulfate of perlecan or bind fibroblasts via surface heparan sulfate proteoglycans. PRELP was first discovered in articular cartilage and highly expressed in cartilage, basement membranes, and developing bone. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-k B inhibitor, however, the effect of PRELP on the differentiation of pre-osteoblastic cell line and the related mechanism has not been reported.Objective:This study aimed to investigate a new role and the mechism of PRELP in modulating osteoblast differentiation. PRELP might be considered as a good choice as an osteopromotive phytomolecule for bone tissue engineering and could also have implications for the treatment of bone diseases in the future.Methods :(1) The study utilized the mouse preosteoblastic MC3T3-E1 cells to establish a cell model of mineralization in vitro. MC3T3-E1 cells were cultured in ostablast inducer medium(growth medium supplemented with MK430) for 21 days, and then the mineralization was evaluated by alizarin red staining, real-time PCR and western-blot.(2) To observe the expression levels of PRELP in different period of osteogenic induction, MC3T3-E1 cells were cultured in ostblast inducer medium. Real-time PCR and western-blot was used to detect the expreeion levels of PRELP.(3) To investigate the impact of PRELP on the differentiation of MC3T3-E1 cells, we used small interfering RNA against expression of PRELP. Real-time PCR and western-blot was used to detect the cut-down effect of PRELP.(4) The effect of PRELP on osteoblastic mineralization was analyzed at the mRNA level of osteogeenic marker genes(Runx2) by real-time PCR. The effects of PRELP on the osteogenic activities in MC3T3-E1 cells were determined by the ALP activity using ALP kit and the mineralized nodules by alizarin red staining.(5) Gene chip was used to investigate the impact of PRELP knockdown on gene expression profile changes in MC3T3-E1 cells.(6) To verify the effects of PRELP on the ?-catenin signal pathway in MC3T3-E1 cells, western-blot was used to detect the total expreeion levels of PRELP, immunofluorescence technique was used to detect the nuclear translocation of ?-catenin in MC3T3-E1 cells, real-time PCR and western-blot was used to detect the expreeion levels of connexin43.Results:(1) Alizarin red staining showed that MC3T3-E1 cells induced by ostablast inducer medium(growth medium supplemented with MK430) produced mineralized nodules, while the control group had no mineralized nodules. Compared with the control, the m RNA expression level of Runx2, ALP and OCN which regulate osteoblast differentiation were upregulated, the expression of Runx2 protein was also increased compared with control.(2) The results of real-time PCR and western-blot showed that the expression of PRELP increased in both mRNA and protein levels after induced by ostablast inducer medium.(3) The results of real-time PCR and western-blot showed that the expression of PRELP increased in both mRNA and protein levels after inhibited the expression of PRELP.(4) Real-time PCR showed that PRELP knockdown could significantly lower the expression of RUNX2, the ALP activity and the formation of mineralized nodules.(5) Gene chip showed that PRELP knockdown dramatically changed the expression profile of MC3T3-E1 cells. The result revealed that ?-catenin gene served as a centered hub gene within this PRELP-related gene network.(6) Western-blot and immunofluorescence technique revealed that PRELP knockdown could significantly lower the total expression and nuclear translocation of ?-catenin in MC3T3-E1 cells, real-time PCR and western-blot revealed that PRELP knockdown inhibited the connexin43 expression in both mRNA and protein levels.Conclusions: Ostablast inducer medium(growth medium supplemented with MK430) induced osteogenic differentiation and mineralization of MC3T3-E1 cells, so we successfully established a cell model of mineralization in vitro. Our results indicated that the expression of PRELP was up-regulated upon stimulation of mineralization in MC3T3-E1 cells. Down-regulation of PRELP expression reduced the capacity of mineralization in MC3T3-E1 cells. Therefore, this study established a new role of PRELP in modulating ?-catenin pathway and osteoblast differentiation.