Department of Genetics and Complex Diseases, Harvard T. H. Chan School of Public Health, Harvard University , Boston, Massachusetts.
Am J Physiol Gastrointest Liver Physiol. 2018 Sep 1;315(3):G351-G363. doi: 10.1152/ajpgi.00103.2018. Epub 2018 May 24.
Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 μM MnCl. However, the hepatocytes were resistant to 4-h exposures of up to 500 μM MnCl despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.
锰（Mn）毒性源于营养问题、社区和职业暴露以及遗传风险。Mn 血液水平受肝胆清除控制。本研究的目的是确定极化肝细胞中 Mn 转运体的细胞分布，建立肝细胞 Mn 外排的体外测定法，并研究 Mn 转运体在金属导入和导出中可能发挥的作用。为此，将 HepG2 细胞培养 12-14 天以获得最大的极性。免疫印迹显示 Mn 转运体 ZIP8、ZnT10、铁蛋白（Fpn）和 ZIP14 存在。间接免疫荧光显微镜将 Fpn 和 ZIP14 定位于 WIF-B 细胞基底外侧区域，而 ZnT10 和 ZIP8 与细胞内囊泡区室相关。ZIP8 阳性结构均匀分布于整个细胞质，但 ZnT10 阳性囊泡与顶端胆区室相邻。WIF-B 细胞对 Mn 毒性敏感，暴露于>250μM MnCl 16 小时后活力下降。然而，尽管 Mn 含量增加了 50 倍，细胞仍能抵抗长达 4 小时高达 500μM MnCl 的暴露。洗脱实验显示，在 4 小时追踪期后，有 80%的 Mn 在时间依赖性的外排中释放。铁调素降低了 WIF-B 细胞中 Fpn 的水平，使 Fpn 从细胞表面清除，但 Mn 外排不受影响。分泌抑制剂布雷菲德菌素 A 阻断了 WIF-B 细胞中 Mn 的释放，提示囊泡融合可能参与了外排。这些结果表明 ZnT10 可能在将 Mn 导入随后与顶膜融合并将其内容物排入胆汁的囊泡中发挥作用。这些结果表明 ZnT10 可能在将 Mn 导入随后与顶膜融合并将其内容物排入胆汁的囊泡中发挥作用。这些结果表明 ZnT10 可能在将 Mn 导入随后与顶膜融合并将其内容物排入胆汁的囊泡中发挥作用。
