Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.
Differentiation. 2011 Feb;81(2):119-26. doi: 10.1016/j.diff.2010.10.003. Epub 2010 Nov 12.
Trichostatin A (TSA) is a histone deacetylase inhibitor (HDACi) known to modulate differentiation of many cells. However, its effect on chondrogenesis remains elusive. This study was aimed to investigate the effects of TSA on in vitro transforming growth factor-β1 (TGF-β1)-induced chondrogenesis of human mesenchymal stem cells (hMSCs). The pellet cultures of hMSCs in a chondrogenic medium were exposed to TGF-β1 and TSA. Quantitative reverse transcription/polymerase chain reaction (PCR) analysis, Alcian blue staining, and immunohistochemistry staining were used to confirm and compare the differences in chondrogenesis by analyzing the mRNA of chondrogenic genes (Sox9, Aggrecan, and Col2A1), synthesis of chondrogenic proteins and type II collagen, respectively. TGF-β1 signaling and its downstream targets were determined by western blot analysis. TGF-β1 led to significant increases in chondrogenic gene expression and the synthesis of chondrogenic proteins. However, TSA significantly decreased chondrogenic gene expression and the synthesis of chondrogenic proteins in a dose-dependent manner. TGF-β1 increased phosphorylation of Smad 2/3 and Sp1 expression around half an hour after induction. The increase of Sp1, but not Smad 2/3 activation was almost completely blocked by the addition of TSA. The chondrogenic effect of TGF-β1 was also suppressed by the Sp1-binding inhibitor mithramycin A. Finally, overexpression of Sp1 abolished TSA-mediated inhibition of TGF-β1-induced chondrogenesis. Our study showed that TSA inhibited chondrogenesis through inhibition of TGF-β1-induced Sp1 expression. Furthermore, Sp1 could be a useful tool in future studies looking into biological mechanisms by which chondrogenesis of hMSCs can be augmented, especially in the area of clinical application.
曲古抑菌素 A（TSA）是一种组蛋白去乙酰化酶抑制剂（HDACi），已知其可调节许多细胞的分化。然而，其对软骨形成的影响仍不清楚。本研究旨在探讨 TSA 对人骨髓间充质干细胞（hMSCs）体外转化生长因子-β1（TGF-β1）诱导软骨形成的影响。在软骨形成培养基中，hMSC 的微球培养物暴露于 TGF-β1 和 TSA。通过分析软骨形成基因（Sox9、Aggrecan 和 Col2A1）的 mRNA、软骨形成蛋白和 II 型胶原蛋白的合成，分别进行定量逆转录/聚合酶链反应（PCR）分析、阿尔辛蓝染色和免疫组织化学染色，以确认和比较软骨形成的差异。通过 Western blot 分析测定 TGF-β1 信号及其下游靶标。TGF-β1 导致软骨形成基因表达和软骨形成蛋白的合成显著增加。然而，TSA 以剂量依赖性方式显著降低软骨形成基因表达和软骨形成蛋白的合成。TGF-β1 在诱导后大约半小时增加 Smad 2/3 的磷酸化和 Sp1 的表达。Sp1 的增加，但不是 Smad 2/3 的激活，几乎完全被 TSA 的添加所阻断。Sp1 结合抑制剂米托蒽醌 A 也抑制了 TGF-β1 的软骨形成作用。最后，Sp1 的过表达消除了 TSA 介导的 TGF-β1 诱导的软骨形成抑制。我们的研究表明，TSA 通过抑制 TGF-β1 诱导的 Sp1 表达抑制软骨形成。此外，Sp1 可能是未来研究 hMSCs 软骨形成的生物学机制，特别是在临床应用领域的有用工具。
