Iemata Mika, Takarada Takeshi, Hinoi Eiichi, Taniura Hideo, Yoneda Yukio
Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan.
J Cell Physiol. 2007 Dec;213(3):721-9. doi: 10.1002/jcp.21145.
Although previous studies including ours have demonstrated the functional expression of different glutamate (Glu) signaling machineries such as Glu receptors (GluRs) and transporters in osteoblasts and chondrocytes, little attention has been paid to the role of Glu in their ancestral mesenchymal stem cells to date. In the present study, we have evaluated the possible functionality of Glu in cultured mouse mesenchymal stem cell line C3H10T1/2 cells endowed to proliferate for the self-renewal and to differentiate toward osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits, in addition to particular excitatory amino acid transporter (EAAT) isoforms and ionotropic GluRs, in undifferentiated C3H10T1/2 cells. Glu significantly suppressed the proliferation activity at a concentration over 500 microM without inducing cell death or differentiation, while the suppression occurred in a manner sensitive to the prevention by cystine and reduced glutathione (GSH), but not by EAAT inhibitors. A significant decrease was seen in intracellular GSH levels in C3H10T1/2 cells cultured with Glu, whereas the cellular proliferation activity was drastically decreased by the addition of the GSH depleter cyclohexene-1-one and the GSH biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Transient overexpression of both xCT and 4F2hc subunits led to an increased basal proliferative activity in C3H10T1/2 cells. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the cystine/Glu antiporter in C3H10T1/2 cells.
尽管包括我们的研究在内的先前研究已经证明了不同谷氨酸（Glu）信号传导机制（如Glu受体（GluRs）和转运体）在成骨细胞和软骨细胞中的功能性表达，但迄今为止，Glu在其祖间充质干细胞中的作用却很少受到关注。在本研究中，我们评估了Glu在培养的小鼠间充质干细胞系C3H10T1/2细胞中的可能功能，这些细胞具有自我更新增殖以及向成骨细胞、软骨细胞、脂肪细胞和肌细胞谱系分化的能力。首次在未分化的C3H10T1/2细胞中显示，除了特定的兴奋性氨基酸转运体（EAAT）亚型和离子型GluRs外，由xCT和4F2hc亚基组成的胱氨酸/Glu反向转运体也有mRNA表达。Glu在浓度超过500微摩尔时显著抑制增殖活性，且不诱导细胞死亡或分化，而这种抑制作用对胱氨酸和还原型谷胱甘肽（GSH）的预防敏感，但对EAAT抑制剂不敏感。在用Glu培养的C3H10T1/2细胞中，细胞内GSH水平显著降低，而分别添加GSH消耗剂环己烯-1-酮和GSH生物合成抑制剂L-丁硫氨酸-[S，R]-亚砜亚胺会使细胞增殖活性急剧下降。xCT和4F2hc亚基的瞬时过表达导致C3H10T1/2细胞的基础增殖活性增加。这些结果表明，Glu可能通过促进C3H10T1/2细胞中胱氨酸/Glu反向转运体的逆向运作，导致细胞内GSH耗竭，从而抑制细胞向自我更新的增殖。
