BMP9-Induced Osteogenic Differentiation of Preadipocytes Through Wnt/β-Catenin and VEGFa Signaling Pathways
WANG Jingjiang, LIU Yang*
The aim of this study is to examine how Wnt/β-catenin and VEGFa (vascular endothelial growth factor a) signaling pathways influence the osteogenic differentiation of preadipocytes induced by BMP9 (bone morphogenetic protein 9). Preadipocytes were infected with AdBMP9 (BMP9 recombinant adenovirus). Western blot analysis was conducted to assess the protein expression levels of β-catenin and VEGFa, while a luciferase reporter assay was utilized to evaluate the activation of the Wnt/β-catenin signaling pathway. After overexpressing or silencing β-catenin and VEGFa, the cells were infected with AdBMP9. ALP (alkaline phosphatase) activity, an early osteogenic marker, was assessed through enzyme activity assays and staining. Western blot was used to detect the protein expression of late osteogenic markers, including OPN (osteopontin), OC (osteocalcin), and the osteogenic transcription factor Runx2 (Runt-related transcription factor 2). Alizarin red staining was used to assess calcium deposition. Ectopic bone formation of preadipocytes in nude mice was examined using Micro-CT and H&E staining. It was found that BMP9 upregulated the protein levels of β-catenin and VEGFa in preadipocytes and increased β-catenin/Tcf4 transcriptional activity. Activation of the Wnt/β-catenin signaling or overexpression of VEGFa promoted BMP9-mediated ALP activity, OPN and OC protein expression, and calcium deposition. Overexpression of β-catenin and VEGFa enhanced BMP9-induced ectopic bone formation in preadipocytes in nude mice. Additionally, overexpression of β-catenin and VEGFa promoted BMP9-induced Runx2 expression, while silencing VEGFa inhibited the BMP9-induced increase in β-catenin/Tcf4 transcriptional activity. These results imply that the Wnt/β-catenin and VEGFa signaling pathways are crucial regulators in the osteogenic differentiation of preadipocytes induced by BMP9.
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