Inhibition of GSK-3β Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2

by Selleckchem | May 29 2023

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3β (GSK-3β), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3β is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3β is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFβ, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of β-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

Comments：

In this study, the authors explored the use of a small molecule inhibitor, 1-Azakenpaullone, to induce osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). The inhibitor specifically targeted glycogen synthase kinase-3β (GSK-3β), a serine-threonine protein kinase that plays a major role in osteoblastic formation by regulating the activity of the transcription factor Runx2.

The authors evaluated the effect of 1-Azakenpaullone on human MSCs using various assays, including alkaline phosphatase activity and staining, Alizarin Red staining, and gene expression profiling using an Agilent microarray platform. The results showed that 1-Azakenpaullone treatment led to increased osteoblastic differentiation and mineralization of human MSCs, as evidenced by higher ALP activity, increased mineralized matrix formation, and upregulation of osteoblast-specific marker gene expression.

Further bioinformatics analysis using Ingenuity Pathway Analysis software identified significant enrichment in genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling, as well as functional categories associated with connective tissue development. The analysis also suggested possible changes in various signaling pathways, including Wnt, TGFβ, and Hedgehog.

Overall, the study suggests that 1-Azakenpaullone could be a promising osteo-promotor factor for bone tissue engineering, as it effectively induced osteoblastic differentiation and mineralization of human MSCs through the activation of Wnt signaling and the upregulation of Runx2.