Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells
Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells
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DOI：
10.1155/2019/3435901
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发表时间：
2019-11-21
影响因子：
4.3
通讯作者：
Alajez, Nehad M.
中科院分区：
文献类型：
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作者：
AlMuraikhi, Nihal;Almasoud, Nuha;Alajez, Nehad M.
背景资料。Hedgehog(HH)信号通路在软骨内成骨过程中对间充质祖细胞的成骨分化起重要作用。然而，HH信号在成人骨重建中的关键作用仍有待阐明。方法：研究方法。在干细胞信号小分子文库的功能筛选中发现一种Smoothed拮抗剂/Hedgehog抑制剂BMS-833923，研究其对人骨骼(间充质)干细胞成骨细胞分化的影响。碱性磷酸酶(ALP)活性和茜素红染色分别作为成骨细胞分化和体外矿化能力的标志。使用Agilent(R)微阵列平台进行全球基因表达谱分析。将hMSC复合羟基磷灰石-磷酸三钙颗粒植入8周龄雌性裸鼠皮下，观察其对体内异位成骨的影响，并用定量组织学方法评价成骨量。结果。SMO拮抗剂/Hedgehog抑制剂BMS-833923对hMSCs的成骨分化有明显的抑制作用，表现为碱性磷酸酶活性的降低、体外矿化和成骨细胞相关基因表达的下调。同样，我们观察到体内异位骨形成减少。与赋形剂处理的对照细胞相比，BMS-833923处理的细胞的全球基因表达谱确定了348个上调和540个下调的基因，这些基因对多个信号通路有显著影响，包括GPCR、软骨内成骨、RANK-RANKL、胰岛素、肿瘤坏死因子α、白介素6和炎症反应。进一步的生物信息学分析表明，在BMS-833923处理的细胞中，涉及结缔组织和骨骼组织发育和疾病的许多功能类别和网络显著丰富，例如，核因子kappaB和STAT信号转导。结论。我们发现SMO/Hedgehog拮抗剂(BMS-833923)是一种有效的抑制人骨髓间充质干细胞成骨分化的药物，可用于治疗与高度异位骨形成和矿化相关的疾病。
Background. Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods. A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent (R) microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results. BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NF kappa B and STAT signaling. Conclusions. We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.