Satoh Yusuke, Matsumura Itaru, Tanaka Hirokazu, Ezoe Sachiko, Sugahara Hiroyuki, Mizuki Masao, Shibayama Hirohiko, Ishiko Eri, Ishiko Jun, Nakajima Koichi, Kanakura Yuzuru
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2004 Jun 11;279(24):24986-93. doi: 10.1074/jbc.M400407200. Epub 2004 Apr 2.
Notch and HOXB4 have been reported to expand hematopoietic stem cells (HSCs) in vitro. However, their critical effector molecules remain undetermined. We found that the expression of c-myc, cyclin D2, cyclin D3, cyclin E, and E2F1 was induced or enhanced during Notch1- or HOXB4-induced self-renewal of murine HSCs. Since c-Myc can act as a primary regulator of G(1)/S transition, we examined whether c-Myc alone can induce self-renewal of HSCs. In culture with stem cell factor, FLT3 ligand, and IL-6, a 4-hydroxytamoxifen-inducible form of c-Myc (Myc/ERT) enabled murine Lin(-)Sca-1(+) HSCs to proliferate with the surface phenotype compatible with HSCs for more than 28 days. c-Myc activated by 4-hydroxytamoxifen augmented telomerase activities and increased the number of CFU-Mix about 2-fold in colony assays. Also, in reconstitution assays, HSCs expanded by c-Myc could reconstitute hematopoiesis for more than 6 months. As for the mechanism of c-myc induction by Notch1, we found that activated forms of Notch1 (NotchIC) and its downstream effector recombination signal-binding protein-J kappa (RBP-VP16) can activate the c-myc promoter through the element between -195 bp and -161 bp by inducing the DNA-binding complex. Together, these results suggest that c-Myc can support self-renewal of HSCs as a downstream mediator of Notch and HOXB4.
据报道，Notch和HOXB4可在体外扩增造血干细胞（HSC）。然而，它们的关键效应分子仍未确定。我们发现，在Notch1或HOXB4诱导的小鼠HSC自我更新过程中，c-myc、细胞周期蛋白D2、细胞周期蛋白D3、细胞周期蛋白E和E2F1的表达被诱导或增强。由于c-Myc可作为G(1)/S转换的主要调节因子，我们研究了单独的c-Myc是否能诱导HSC自我更新。在含有干细胞因子、FLT3配体和IL-6的培养体系中，一种4-羟基他莫昔芬诱导型的c-Myc（Myc/ERT）能使小鼠Lin(-)Sca-1(+) HSC以与HSC相容的表面表型增殖超过28天。4-羟基他莫昔芬激活的c-Myc增强了端粒酶活性，并在集落试验中使CFU-Mix数量增加了约2倍。此外，在重建试验中，由c-Myc扩增的HSC能在超过6个月的时间里重建造血功能。至于Notch1诱导c-myc的机制，我们发现激活形式的Notch1（NotchIC）及其下游效应分子重组信号结合蛋白-Jκ（RBP-VP16）可通过诱导DNA结合复合物，激活-195 bp至-161 bp之间元件的c-myc启动子。总之，这些结果表明，c-Myc可作为Notch和HOXB4的下游介质支持HSC的自我更新。
