Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK
Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK
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DOI：
10.1074/jbc.ra119.010329
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发表时间：
2019-11
期刊：
影响因子：
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通讯作者：
S. Wakabayashi;Hirofumi Morihara;Shunichi Yokoe;T. Nakagawa;Kazumasa Moriwaki;K. Tomoda;M. Asahi
中科院分区：
文献类型：
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作者：
S. Wakabayashi;Hirofumi Morihara;Shunichi Yokoe;T. Nakagawa;Kazumasa Moriwaki;K. Tomoda;M. Asahi
了解人类诱导多能干细胞（iPSCs）的特定特性对于iPSCs的质量控制至关重要。我们偶然发现质膜Na⁺/H⁺交换体1（NHE1）的过表达会诱导iPSCs死亡，于是研究了NHE1诱导细胞死亡的机制。多西环素诱导的NHE1过表达使细胞生长停滞，并且几乎所有细胞在72小时内都通过坏死过程死亡。NHE1过表达导致Rho相关卷曲螺旋激酶（ROCK）持续激活，同时iPSC集落中的细胞形状、细胞伸长以及周边细胞肿胀发生显著变化，并且应力纤维形成明显。ROCK抑制剂Y27632减少了NHE1诱导的细胞死亡。NHE1的功能缺失突变抑制了ROCK依赖的表型，NHE1活性抑制剂也抑制了这些表型，这表明需要NHE1介导的转运活性。此外，三甲胺处理介导的胞质碱化激活了ROCK，并且ROCK在细胞集落周边的iPSCs中在靠近NHE1的质膜处积累。相比之下，从iPSCs分化而来的中内胚层样细胞没有发生细胞死亡，这表明NHE1介导的效应是iPSCs所特有的。这些结果表明，NHE1过表达通过持续的ROCK激活特异性地诱导iPSCs死亡，这可能是由NHE1附近局部pH升高引起的。最后，莫能菌素（一种Na⁺/H⁺交换离子载体）选择性地杀死iPSCs，这表明莫能菌素有助于消除分化后残留的iPSCs，这一策略可能对改进再生医学有用。
Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment–mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.