The Effect Of Sirt2on The Process Of Osteoblast Differentiation

Runx2and Osterix are the two important transcription factors, which play pivotalroles in osteoblast differentiation. The gene expression and activities of Runx2andOsterix during osteoblastic differentiation from mesenchymal stem cells are not onlyconstantly regulated by extracellular signals and intracellular signaling pathways, butalso regulated by other factors and through interactions with other factors. Manytranscription factors, growth factors and hormones are reported to affect theexpression and activities of Runx2, whereas many Runx2interacting proteins are alsoemerging as Runx2regulators. For example, some histone deacetylase proteins(HDACs) interact with Runx2and regulate Runx2activities and thereby boneformation.On the basis of the previous studies on Runx2, HDACs and their interactions byothers, we further studied on the effects of Sirt2on the process of osteoblastdifferentiation. First, we examined the endogenous protein levels of Sirt2in a varietyof cell lines in osteoblastic lineage, and then generated various stable cell lines ofoverexpressing Sirt2in C3H10T1/2whose nature is a mesenchymal stem cell line.The results showed weak expression of Sirt2in the rat osteosarcoma cells ROS17/2.8,preosteoblasts MC3T3-E1, osteocytes MLO-Y4, mesenchymal stem cells C3H10T1/2,and high expression of Sirt2in the Sirt2overexpressing cell lines. Luciferase reporterassay revealed that in the undifferentiated condition. Sirt2repressed Runx2mediatedactivation of OCN promoter, after48h osteoblastic differentiation, Sirt2still repressedRunx2mediated activation of OCN promoter. In this study Osterix did not affect theOCN promoter activity, we failed tot analyze whether Sirt2represses Osterixmediated activation of OCN promoter. RT-PCR and Alkaline phosphatase (ALP)activity staining demonstrated that Sirt2did not affect ALP expression, but may affectthe Runx2mediated regulation of OCN gene expression, That Sirt2represses Runx2mediated OCN promoter activity and gene expression implyed that Sirts may play anegative role in the late stage osteogenic differentiation.By using immuno-fluorescence technique, we next found that Runx2and Osterixmainly localized in the nucleus, while Sirt2mainly localized in the cytoplasm, bothendogenous and exgenous. This phenotype prompts us to suppose that the Sirt2effects on Runx2or Osterix may be in an indirect manner, rather than in a directmanner. The specific indirect mechanism needs further study. In the mean time, because Sirt2can inhibit mitotsis, we next examined whether Sirt2affects osteoblastproliferation. According to the results from direct cell counting, we drawed the growthcurve and found that Sirt2inhibited the proliferation of the Runx2overexpressingcells. Therefore, we propose that Sirt2may inhibit cell proliferation at an early stageof osteoblastic differentiation, although Sirt2does not affect the ALP activity, which isa representative marker for a early stagen osteoblastic differentiation.