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Objective To investigate the effect of bone morphogenetic protein 4 (BMP4) overexpression in MLO-Y4 osteocytes on osteogenic differentiation of bone marrow stromal ST2 cells through Notch signaling. Methods Ad-GFP or Ad-BMP4 infected MLO-Y4 cells were co-cultured with ST2 cells, and designated as GFP and BMP4 group respectively. Western blotting was used to detect the BMP4 protein level in MLO-Y4 cells. Real-time fluorescence quantitative analysis (qPCR) was applied to determine the mRNA expression of BMP4 and Notch ligands genes as well as osteoblast genes in the MLO-Y4 cells, and Notch target genes and angiogenesis factors in the co-cultured systems. The activity of alkaline phosphatase (ALP) was determined by ALP staining and biochemical quantification. Notch signal inhibitor DAPT was added to the co-culture system to detect the above indexes again. Results Overexpression of BMP4 upregulated the expression of Notch ligands Jag1, Jag2 and Dll4 in MLO-Y4 cells. ALP staining and biochemical quantitative results demonstrated that there was no significant difference in ALP activity at day 1 between the GFP and BMP4 groups, and stronger ALP activity was seen in the latter group at day 3 and 5 (P < 0.05). In the BMP4 group, at day 1 among the osteoblast genes, only runt-related transcription factor 2 (Runx2) was obviously expressed (P < 0.05), and the levels of Alp, Runx2, osteocalcin (Ocn), and collagen Ⅰ were all highly expressed in the BMP4 group (P < 0.05). At day 3, Notch signaling target genes Hey1 and Hey2, angiogenesis factors vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF1α) were also highly expressed in the BMP4 group compared with the control group (P < 0.05). After addition of DAPT to inhibit Notch signaling, the activity of ALP and expression of osteoblast genes and angiogenesis genes were all decreased (P < 0.05). Conclusion Overexpression of BMP4 in MLO-Y4 cells promotes osteogenic differentiation of ST2 cells, which may be related to the enhancement of Notch signaling. |
