Department of Pathology and Laboratory Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, 02912, USA.
Vor Biopharma, Cambridge, MA, USA.
Biometals. 2021 Jun;34(3):573-588. doi: 10.1007/s10534-021-00296-y. Epub 2021 Mar 13.
Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of Mn import and export in mutant cells. While impaired Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.
锰（Mn）是一种必需的金属，在高水平下可能有毒。2012 年，首次报道了 SLC30A10 基因突变的患者存在锰过量的遗传原因，SLC30A10 是一种锰外排转运体。为了体外研究 SLC30A10 的功能，本研究使用 CRISPR/Cas9 基因编辑技术开发了稳定的 SLC30A10 突变 Hep3B 肝癌细胞系，并在活小鼠中使用胶原酶灌注分离 Slc30a10 缺失的原代肝细胞。我们还比较了原代和非原代细胞系的表型，以确定它们是否都可以作为 SLC30A10 已知生理功能的可靠体外模型。暴露于过量锰时，突变 SLC30A10 Hep3B 细胞的锰水平升高，活力降低。转运研究表明突变细胞中锰的输入和输出减少。虽然鉴于 SLC30A10 在细胞内锰输出中的重要作用，推测锰外排受损，但锰内流受损是出乎意料的。全基因组测序未在突变 Hep3B 突变细胞系中发现已知锰转运体的任何其他突变。然后，我们评估了从具有不同肝脏锰水平的基因改变小鼠中分离的原代肝细胞培养物中的锰转运。基于这些实验的结果，我们建议可以在体外研究 SLC30A10 缺乏对锰稳态的影响，但仅在特定类型的细胞系中。
