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

• Published in: International journal of molecular sciences 2023-04, Vol.24 (8), p.7164
• Main Authors: AlMuraikhi, Nihal, Binhamdan, Sarah, Alaskar, Hanouf, Alotaibi, Amal, Tareen, Sumaiya, Muthurangan, Manikandan, Alfayez, Musaad
• Format: Article
• Language: English
• Subjects: Analysis; beta Catenin - metabolism; Cell Differentiation - genetics; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; Development and progression; Genes; Genetic transcription; Glycogen; Glycogen Synthase Kinase 3 beta - genetics; Glycogen Synthase Kinase 3 beta - metabolism; GSK-3β inhibition; human mesenchymal stem cells; Humans; Mesenchymal Stem Cells - metabolism; osteoblast differentiation; Osteoblasts - metabolism; Osteogenesis - genetics; Phosphatases; Photographic industry; Protein kinases; Runx2; Stem cells; Synthesis; Tissue engineering; Transforming growth factors; Type 2 diabetes; Wnt; Wnt Signaling Pathway - physiology
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24087164

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.