Misumi Sachiyo, Kim Tae-Sun, Jung Cha-Gyun, Masuda Tadashi, Urakawa Susumu, Isobe Yoshiaki, Furuyama Fujiya, Nishino Hitoo, Hida Hideki
Department of Neurophysiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan.
Eur J Neurosci. 2008 Sep;28(6):1049-59. doi: 10.1111/j.1460-9568.2008.06420.x. Epub 2008 Sep 9.
We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.
我们之前已经证明，一种G1/S期细胞周期阻滞剂去铁胺（DFO）可增加大鼠神经球培养物中新神经元的数量，这与细胞周期蛋白依赖性激酶（cdk）抑制剂p27（kip1）的延长表达相关[H. J. Kim等人（2006年）《脑研究》，1092，1 - 15]。本研究聚焦于用G1/S期细胞周期阻滞剂处理神经干/祖细胞（NPCs）后的神经元分化机制。向神经球中添加DFO（0.5 mM）或阿非科林（Aph）（1.5 μM）8小时，随后进行3天的分化，导致神经元数量增加和神经突生长。DFO在分化后24小时诱导转化生长因子（TGF）-β1和cdk5表达增强，而Aph仅增加TGF-β1表达。给予cdk5抑制剂罗司维汀可减弱DFO诱导的神经发生和神经突生长，表明DFO和Aph的神经发生机制不同。TGF-β1（10 ng/mL）并未增加神经突生长，而是增加了β-微管蛋白III阳性细胞的数量，这伴随着p27（kip1）mRNA表达增强。此外，在巢蛋白阳性的NPCs中观察到II型TGF-β受体表达。结果表明，DFO诱导的TGF-β1信号激活了smad3从细胞质向细胞核的转位。相反，通过I型TGF-β受体抑制剂（SB - 505124）抑制TGF-β1信号，导致DFO诱导的神经发生减少，同时p27（kip1）蛋白表达和smad3向细胞核的转位也减少。这些结果表明，G1/S期的细胞周期停滞诱导TGF-β1表达。这反过来又通过smad3向细胞核的转位以及随后NPCs中p27（kip1）的激活促进神经元分化增强。
