Rapamycin Promotes the Osteoblastic Differentiation of Human Embryonic Stem Cells by Blocking the mTOR Pathway and Stimulating the BMP/Smad Pathway
Rapamycin Promotes the Osteoblastic Differentiation of Human Embryonic Stem Cells by Blocking the mTOR Pathway and Stimulating the BMP/Smad Pathway
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DOI：
10.1089/scd.2009.0147
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发表时间：
2010-04-01
影响因子：
4
通讯作者：
Cho, Yee Sook
中科院分区：
文献类型：
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作者：
Lee, Kyu-Won;Yook, Jin-Yong;Cho, Yee Sook
研究发现，PI3K/AKT/mTOR信号通路在人胚胎干细胞（human embryonic stem cell, hESC）自我更新和分化的调控中起重要作用。然而，其对hESCs成骨分化的作用尚不清楚。我们测试了药理学PI3K/AKT/mTOR抑制剂对hESCs诱导成骨分化的影响。在无饲料培养条件下，雷帕霉素（一种mTOR抑制剂）能有效抑制未分化hESCs中mTOR和p70S6K的活性；然而，LY294002 （PI3K抑制剂）和AKT抑制剂没有影响。这些抑制剂均可下调hESC标志物Oct4和Nanog，但只有雷帕霉素可诱导早期成骨标志物BMP2和Runx2上调。我们还观察到，用雷帕霉素的结构类似物FK506处理hESCs分化，但不表现出成骨表型。Smad1/5/8磷酸化和Id1-4 mRNA表达的增加表明雷帕霉素显著刺激了BMP/Smad信号传导。用雷帕霉素诱导hESCs和人胚状体（hEBs） 2-3周后，成骨细胞分化进一步通过成骨细胞标志物mrna和/或蛋白（骨钙素、骨保护素、骨连接素和骨涎蛋白）的表达、碱性磷酸酶活性和矿化骨结节形成的茜素红S染色来表征。雷帕霉素分化的hESCs和hEBs在成骨表型上无显著差异。我们的研究结果表明，在这3种抑制剂中，只有雷帕霉素是hESCs成骨细胞分化的有效刺激因子，它是通过调节雷帕霉素敏感的mTOR和BMP/Smad信号来实现的。
Studies revealed that PI3K/AKT/mTOR signaling is important in the regulation of human embryonic stem cell (hESC) self-renewal and differentiation. However, its action on osteogenic differentiation of hESCs is poorly understood. We tested the effects of pharmacological PI3K/AKT/mTOR inhibitors on their potential to induce osteogenic differentiation of hESCs. Under feeder-free culture conditions, rapamycin (an mTOR inhibitor) potently inhibited the activities of mTOR and p70S6K in undifferentiated hESCs; however, LY294002 (a PI3K inhibitor) and an AKT inhibitor had no effects. Treatment with any of these inhibitors down-regulated the hESC markers Oct4 and Nanog, but only rapamycin induced the up-regulation of the early osteogenic markers BMP2 and Runx2. We also observed that hESCs differentiated when treated with FK506, a structural analog of rapamycin, but did not exhibit an osteogenic phenotype. Increases in Smad1/5/8 phosphorylation and Id1-4 mRNA expression indicated that rapamycin significantly stimulated BMP/Smad signaling. After inducing both hESCs and human embryoid bodies (hEBs) for 2-3 weeks with rapamycin, osteoblastic differentiation was further characterized by the expression of osteoblastic marker mRNAs and/or proteins (osterix, osteocalcin, osteoprotegerin, osteonectin, and bone sialoprotein), alkaline phosphatase activity, and alizarin red S staining for mineralized bone nodule formation. No significant differences in the osteogenic phenotypes of rapamycin-differentiated hESCs and hEBs were detected. Our results suggest that, among these 3 inhibitors, only rapamycin functions as a potent stimulator of osteoblastic differentiation of hESCs, and it does so by modulating rapamycin-sensitive mTOR and BMP/Smad signaling.