The Study Of Role Of DDR2 In Osteoblast Differentiation

Osteoblasts differentiation involves a series of well orchestrated processes that are regulated by many effectors and factors. The interaction between osteoblasts and their extracellular matrix is of great importance to osteoblasts differentiation. In this so-called dialogue, the sensor receptors on the surface of osteoblasts receive the stimulating signal from extracellular environment, response to the signal and start a series of signaling cascade, to promote osteoblasts differentiation.α2β1 integrin is one of this sensor on the osteoblasts surface, it response to the stimulating signal from the outside of the cells, and play an important role in osteoblasts differentiation. Upon the simulation of collagen I,α2β1 integrin regulate the expression of osteoblastic gene marker though modulating Runx2 transcriptional activity, to take function in osteoblasts differentiation.Besides the integrin family, DDRs family is another collagen I receptor. One of the most important phenotypes of Ddr2 knockout mice is shortening of long bones. Mutation in Ddr2 cause human SMED with puffy hands. DDRs is a kind of receptor tyrosine kinase, and is named by their discoidin-like domain. DDR1 can be activated by most collagen, whereas DDR2 can only be activated by fibrous collagen, particularly by type I and III collagen.Based on these results above, we speculate wether DDR2, functioning as recpters for collgen I, takes fuction in osteoblasts differentiation asα2β1 integrin? To date, there is no report about DDR2's fuction in osteoblasts differentiation. Thus, we begin to research the role of DDR2 in Osteogenesis.First, we established three kind of osteoblasts differentiation model, including mouse preosteoblast MC3T3-E1 model, mouse premyocyte C2C12 model and primary mouse BMSC model. We detect the expression pattern of DDR2 in the process of osteoblasts differentiation. We found that the expression of DDR2 increased in the early period, and gradually decrease until to the basal level after fully differentiated. However, the expression of DDR1 was not alterd. The phosphorylation analysis showed that the DDR2 was activated after cell fully differentiated. So we focused on the DDR2.In order to define the role of DDR2 in osteoblastes differentiation, we generated stable clones in which DDR2 expresssion is downregulated or upregulated using lentivirus. We analyzed the difference of differentiation speed between control cells and genetically modified cells, by detecting the mRNA expression of ALP/OCN, the activity of ALP, the production of OCN and the formation of mineralized nodules. We found that downregulation of DDR2 caused delayed osteoblastes differentiation, whereas upregulation of DDR2 accerlerated osteoblastes differentiation.To further investigated the molecular mechanism of DDR2's role in osteoblastes differentiation, we tested the change of Runx2 transcriptional activity after DDR2 was modified. Though analyzing of OCN (Runx2 target gene) promoter activity, OSE2 luciferase activity and EMSA, we found that deletion of DDR2 decreased the Runx2 transcriptional activity, whereas acivition of DDR2 elevated Runx2 transcriptional activity. DDR2 promote osteoblasts differentiation though modulating Runx2 transactivity.By analyzing the difference of Runx2 expression and subcellular localization, we found that neither Runx2 expression level and subcellular localization was not effected by DDR2, whereas Runx2 phosphorylation was influenced by DDR2. DDR2 sh RNA expressiong cells showed lower Runx2 phosphryation level under differentiated condition. FcDDR2 expressing cells showed higher Runx2 phosphoryation under both un differentiated and differentiated conditions. The phosphoryation of Runx2 caused by collagen I is also changed in this manner.It is reported that ERK MAPK plays an essential role in bone development and phosphorylation of Runx2. ERK is also involved in DDR2-induced MMP13 expression. Whether ERK participates in the regulation of Runx2 transactivity caused by DDR2?Fisrst we analyzing the ERK expression pattern during osteoblasts differentiation. We found that the phophorylation of ERK was significantly decreased in DDR2 shRNA expressing cells. Then we employed PD98059, an specific ERK inhibitor, to tested the differnence of Runx2 transcriptional activity induced by FcDDR3 between control cells and genetically modified cells. The results showed that, the transactivity of Runx2 induced by DDR2 activation was significantly suppressed in DDR2 shRNA expressing cells. To futher verify the ERK's involvement, we do rescue experiment using Runx2 mutant plasmids. We found that the down regulation of Runx2 transactivity and maker gene expression were more or less restored.In this study, we suppose a DDR2's important role in osteblasts differentiation, and a possible molecular mechanism. We supply a new prospect for the study of osteoblasts differnentiation and update the signaling pathway of DDR2.