Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia.
Int J Mol Sci. 2023 Apr 12;24(8):7164. 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.
小分子抑制剂诱导的成骨分化已被广泛应用于调控成骨相关信号通路。在本研究中，我们鉴定出 1-氮杂 kenpaullone（一种高度选择性的糖原合酶激酶-3β（GSK-3β）抑制剂）是一种强有力的人骨髓间充质干细胞（hMSCs）成骨分化和矿化诱导剂。GSK-3β 是一种丝氨酸/苏氨酸蛋白激酶，在多种疾病的发展中起着重要作用。GSK-3β 是成骨形成中 Runx2 活性的关键调节因子。我们通过碱性磷酸酶活性和染色试验评估成骨细胞分化，通过茜素红染色评估培养的 hMSCs 的矿化。使用安捷伦微阵列平台评估基因表达谱，并使用 Ingenuity Pathway Analysis 软件进行生物信息学分析。用 1-氮杂 kenpaullone 处理的 hMSCs 显示出更高的碱性磷酸酶活性、体外矿化基质形成增加，以及成骨特异性标记基因表达上调。与对照细胞相比，1-氮杂 kenpaullone 处理的 hMSCs 的全基因表达谱分析鉴定出 1750 个上调和 2171 个下调的 mRNA 转录本。这也表明各种信号通路可能发生变化，包括 Wnt、TGFβ 和 Hedgehog。进一步采用 Ingenuity Pathway Analysis 的生物信息学分析，在 1-氮杂 kenpaullone 处理的细胞中发现与 cAMP、PI3K（复合物）、P38 MAPK 和 HIF1A 信号转导相关的遗传网络以及与结缔组织发育相关的功能类别显著富集。我们的结果表明，1-氮杂 kenpaullone 通过激活 Wnt 信号和β-catenin 的核积累显著诱导 hMSCs 的成骨分化和矿化，从而上调 Runx2，这是一种关键的转录因子，最终促进成骨特异性基因的表达。因此，1-氮杂 kenpaullone 可用作骨组织工程中的成骨促进因子。
