Sabelli Manuela, Montosi Giuliana, Garuti Cinzia, Caleffi Angela, Oliveto Stefania, Biffo Stefano, Pietrangelo Antonello
Division of Internal Medicine 2 and Center for Hemochromatosis, University Hospital of Modena, Modena, Italy.
INGM, 'Romeo ed Enrica Invernizzi', Milano, Italy.
Hepatology. 2017 May;65(5):1512-1525. doi: 10.1002/hep.29007. Epub 2017 Mar 22.
Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention.
FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).
铁转运蛋白1（FPN1）是哺乳动物中唯一的铁输出蛋白，但其细胞特异性功能和调节机制仍不清楚。本研究检测了人巨噬细胞中铁转运蛋白1的表达情况，这类细胞主要负责日常血浆铁周转，并且在铁转运蛋白病（FD）的发病机制中起核心作用，FD是一种由功能缺失性FPN1突变引起的疾病。我们通过共聚焦显微镜、蛋白质免疫印迹、凝胶过滤和免疫沉淀研究，对来自对照献血者（献血者）以及携带FPN1 p.A77D、p.G80S、p.Val162del功能缺失或p.A69T功能获得性突变的患者的巨噬细胞中的FPN1蛋白表达和运输进行了表征。我们发现，在正常巨噬细胞中，早期内吞小室中的FPN1循环单体不会多聚化，并在3 - 6小时内被其生理抑制剂铁调素（Hepc）迅速降解。在FD巨噬细胞中，内源性FPN1表现出相似的定位，只是在溶酶体中的积累更多。然而，与之前在细胞系中使用过表达突变蛋白的研究不同，FPN1仍能到达细胞表面，并在暴露于Hepc后正常内化和降解。但是，当FD巨噬细胞暴露于大量血红素铁时，与献血者和p.A69T巨噬细胞不同，FPN1不再能到达细胞表面，导致细胞内铁潴留。
FPN1在内吞/质膜区室中作为单体循环，并在对照细胞和FD细胞中对其生理抑制剂Hepc作出反应。然而，在FD中，当细胞经历高铁周转时，FPN1无法到达细胞表面。我们的研究结果为FD的特征提供了依据，FD的特征是肠细胞（即铁周转低的细胞）中的铁转运得以保留，而暴露于高铁通量的细胞（如肝脏和脾脏巨噬细胞）中则出现铁潴留。（《肝脏病学》2017年；65:1512 - 1525）
