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  • The purposes of this study are to


    The purposes of this study are to investigate the molecular function of DDR2, especially in cellular proliferation in prechondrocytes, and to examine the relationship between the proliferation and differentiation of prechondrocytes through both in vitro and in vivo analysis.
    Materials and methods
    Discussion Endochondral proliferation and ossification are very important but complex processes for bone and skeletal formation, which is controlled by the spatial and temporal environments. To date, little is known about the exact role of extracellular matrix molecules, specifically collagen and its receptors, in endochondral ossification. DDR2 null allele dwarf mice have chondrocyte abnormalities [11], along with four different mutations in the DDR2 gene in the patients affected with the rare autosomal-recessive spondylo-meta-epiphyseal dysplasia, short limb-abnormal calcification-type syndrome (SMED-SL) [32], [33]. However, the details of DDR2 regulation about ligands, molecules, receptors, and intracellular signaling pathways of the extracellular matrix involved in endochondral proliferation and ossification are not well known. Here we used the ATDC5 cell line as a model of endochondral proliferation and ossification. The miDdr2-transfected ATDC5 cell lines are useful models of precursor endochondral chemical informative post because we found that they retained normal differentiation ability. DDR2 reduction induced by miRNA designed for Ddr2 finally promoted cellular proliferation in proportion to the decrease in the ratio of Ddr2 expression, indicating that DDR2 might play a negative role in the proliferation of chondrocytes. We also identified that miDdr2-transfected ATDC5 cell lines showed earlier differentiation to cartilage cells by insulin induction compared with mock cells after 15d culture. This study suggested that DDR2 might play an inhibitory regulatory role on chondrocyte differentiation. Taken together, our results indicate that the decrease in DDR2 leads to cellular proliferation due to the direct signal reduction of DDR2, not to the loss of function of endochondral cells in miDdr2-transfected ATDC5 cells. DDR2 activates Runx2 through p38 MAPK to promote osteoblast differentiation [34]. We demonstrated that reduced expression of DDR2 may lead to the subsequent downregulation of Runx2 in miDdr2-transfected ATDC5 cells in proportion to the expression reduction level of Ddr2. This result indicated that Runx2 could be a downstream molecule of the Ddr2 signal pathway in endochondral cells. DDR2 acts as a proliferative regulator to fibroblast and hepatic stellate cells through the extracellular matrix [11], [13], [27]. These results and our results may contradict each other, but there are two possibilities for explaining the discrepancy. First is the decrement of Runx2 expression in miDdr2-transfected ATDC5 cells. Runx2 is essential for chondrocyte maturation. It regulates limb growth by organizing chondrocyte maturation and proliferation and by repressing adipogenic differentiation and maintaining the cellular character of chondrocytes in vitro[35], [36], [37]. In miDdr2-transfected ATDC5 cells, the reduction of Runx2 expression according to the DDR2 decrement resulted in consecutively proliferative cellular induction by maintaining the undifferentiated status of chondrocytes. Secondly, the reduction of DDR2 expression is systemic in Ddr2 knockout mice and spontaneous mutant mice, and dwarfism also can be provoked by reasons other than only the decrease in the ability of chondrocyte cellular proliferation. In fact, DDR2 deletion yielded many phenotypes, including pituitary and reproductive functions [12]. Further research for DDR2 should be conducted, as our results did not reflect the temporal or partial ossification situation because chemical informative post osteoblasts and osteoclastic cells are dimensionally involved in endochondral ossification [24], [25]. Next, we tried to specifically inhibit DDR2 function in endochondral proliferation and ossification in vivo. We used competitive inhibition with KD-Ddr2 overexpression specifically in cartilage cells, because it is generally difficult to regulate genes by miRNA in vivo. We constructed a transgenic vector using an insulator sequence to stabilize the activity of the transgene expression [30]. Body size and skeleton length of KD-Ddr2 overexpressed mice were not significantly different compared with the littermates. On the other hand, the layer of hypertrophic chondrocytes in KD-Ddr2 transgenic mice was not significantly thicker than that of normal littermates, but the layer of proliferative chondrocytes in KD-Ddr2 transgenic mice was significantly thicker than that of normal littermates. This cellular proliferation is similar to the phenotype of miDdr2-transfected ATDC5 cells in vitro, which indicated that DDR2 might have various molecular functions to regulate the proliferation of chondrocytes rather than the differentiation of hypertrophic chondrocytes.