Effect of Activation of Wnt Microenvironment of MLO-Y4 Osteocytes in Co-Culture System on Osteogenic Differentiation of ST2 Bone Marrow Stromal Cells
WANG Bo1, LUO Yisheng2, LI Zhongjun1
The aim of this article was to investigate the effects of activation of the Wnt microenvironment of osteoblast MLO-Y4 by the small molecule drug C91 (CHIR99021) on the osteogenic differentiation of ST2 cells after co-culturing the treated osteoblast MLO-Y4 cells (without C91) with bone marrow stromal cells ST2 cells. CCK8 assay was performed to detect the effects of different concentrations of C91 on osteoblastic MLO-Y4 activity and to determine the concentration of C91 used for subsequent experiments; ALP (alkaline phosphatase) staining, ARS (alizarin red S) staining and quantitative analysis were used to detect the level of osteogenic differentiation; fluorescence quantitative PCR was used to detect the mRNA expression levels of the ST2 cell osteogenic differentiation marker genes Alp, Runx2, Osx, the vasculogenic factor Vegf, and the Wnt signaling target genes β-catenin, Axin2 and Lef1, as well as the osteogenic inhibitory factor Sost; immunofluorescence staining detected the entry of β-catenin into the nucleus; Western blot detected the protein expression level of β-catenin in the cells. When the concentration of C91 was 2.5 μmol/L and 5 μmol/L, C91 had no effect on the proliferative activity of osteoblast MLO-Y4 regardless of the action time of 12 h and 24 h, which can be used as the maximum safe con centration for the subsequent experiments; C91 promoted the mRNA expression of the Wnt signaling target genes β-catenin, Axin2, Lef1, and osteoclastogenic factor Sost in a dose-dependent manner (P<0.05), and the expression level of β-catenin protein was up-regulated (P<0.05); C91 was able to significantly promote the nucleation process of β-catenin in osteoblasts MLO-Y4; following activation of the Wnt microenvironment in the co-culture system of bone cells MLO-Y4 by C91, the mRNA expression levels of Alp, Runx2, Osx, and the angiogenic factor Vegf in ST2 cells increased in a dose-dependent manner (P<0.05), however, the expression level of the osteogenic differ entiation factor Ocn was significantly reduced (P<0.05). C91 treatment promotes early osteogenic differentiation of ST2 cells in co-culture systems (as evidenced by elevated alkaline phosphatase expression), while simultaneously inhibiting their late-stage matrix mineralization.
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