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  • br Materials and methods br


    Materials and methods
    Discussion Our previous study showed that ERRγ negatively regulates osteoblast differentiation via inhibiting Runx2 transactivity (Jeong et al., 2009). In the present study, we found that ERRγ also induced miR-433 in osteogenic mesenchymal cell lineage C3H10T1/2 cells, and inhibited BMP2-induced osteoblastic differentiation via direct targeting of inducible miR-433 to 3′-UTR region of Runx2 mRNA. This study provides evidence that ERRγ inhibits BMP2-induced osteoblastic differentiation of MSC via a post-transcriptional modification of Runx2. In the present study, RT-PCR and real-time PCR analysis showed that the ERRγ and miR-433 were highly expressed in undifferentiated mesenchymal stem Ketanserin and and their expressions were decreased under osteogenic stimuli such as BMP2 treatment. These results suggest that the highly expressed ERRγ is involved in maintaining the undifferentiated status of mesenchymal stem cell via miR-433-mediated inhibition of osteogenic factors such as Runx2. On the other hand, in our previous studies using pre-osteoblasts and primary osteoblasts, BMP2 treatment increased ERRγ expression up to 2days and decreased after that (Jeong et al., 2009). The findings showed that ERRγ responses to BMP2 seem to vary depending on type of cells or degree of cell differentiation. In fact, pre-osteoblast MC3T3E1 and primary calvarial osteoblasts are known to be committed to differentiated status from progenitor cells by some osteogenic factors, whereas C3H10T1/2 cells were reported to keep their mesenchymal stem cell traits, which can differentiate into osteoblast, chondrocyte, and adipocyte. The function of miR-433 was recently reported in hepatocytes (Song and Wang, 2008). They demonstrated that ERRγ increased miR-433 expression via direct binding to ERRE site on the miR-433 promoter region. Recently, several studies have reported that microRNAs are involved in osteoblast differentiation (Eskildsen et al., 2011, Hu et al., 2011, Li Ketanserin and et al., 2009, Zhang et al., 2011). Especially, the miR-3960 and miR-2861 can control osteoblast differentiation via feedback loop with Runx2 (Hu et al., 2011). Runx2 is a major transcription factor in the early stage of osteoblast differentiation. Runx2 regulates the expression of several osteogenic genes, including collagen type I, osteopontin, osteocalcin and bone sialoprotein (Ducy et al., 1999, Javed et al., 2001), by binding to the osteoblast-specific element (OSE) of their genes (Ducy and Karsenty, 1995). In the present study, we found that miR-433 suppresses Runx2 expression via direct binding to S1 and S2 sites on 3′ UTR of Runx2 in C3H10T1/2 cells. In addition, ERRγ-inhibited Runx2 expression was recovered by anti-miR-433. These findings suggest that miR-433 could control the osteoblastic differentiation of mesenchymal stem cells through the modulating Runx2, and that Runx2 may be regulated by various miRNAs, including miR-3960, miR-2861, and miR-433 in osteoblast cells. In addition, action of miRNAs in regulation of Runx2 seems to be diverse. For example, the miR-3960 and miR-2861 increase Runx2 expression indirectly, but miR-433 suppresses Runx2 expression by direct binding to 3′ UTR region. In summary, ERRγ induction of miR-433 decreases level of Runx2 transcript by directly binding to 3′ UTR of Runx2 in C3H10T1/2 cells, and inhibits osteoblastic differentiation of mesenchymal stem cells. In the osteogenic differentiation state with BMP2, ERRγ and miR-433 were decreased, and induced the expression of osteogenic genes via an increase in Runx2 expression (Fig. 4E). These observations suggest that miR-433 has an important role in osteoblastic differentiation of mesenchymal stem cells, and that anti-miR-433 may be used as a therapeutic agent for the treatment of bone-related diseases.
    Conflict of interest statement
    Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MEST) to J.T.K. (No. 2011-0030759), and W.G.J. was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education, Science and Technology (2012R1A1A2001408).