Wallace Daniel F, Summerville Lesa, Crampton Emily M, Frazer David M, Anderson Gregory J, Subramaniam V Nathan
Membrane Transport Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia.
Hepatology. 2009 Dec;50(6):1992-2000. doi: 10.1002/hep.23198.
Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2; Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin.
These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8.
铁调素是铁稳态的核心调节因子。HFE和转铁蛋白受体2（TFR2）在成年发病型遗传性血色素沉着症中发生突变，并根据铁含量调节铁调素的表达。它们是通过相同途径还是平行途径发挥作用尚不清楚。为了研究这一点，我们通过在基因相同的背景下将Hfe和Tfr2基因敲除小鼠杂交，构建了同时缺失Hfe和Tfr2基因的小鼠模型。对野生型、单基因敲除和双基因敲除小鼠的组织和血清进行了分析。测定了血清转铁蛋白饱和度和肝脏铁浓度。通过实时聚合酶链反应（PCR）分析铁相关信使核糖核酸（mRNA）转录本的表达。通过免疫印迹分析铁相关蛋白Tfr1、Tfr2、铁蛋白和铁调素原的水平，以及细胞信号蛋白细胞外信号调节激酶1/2（Erk1/2）和Smad1/5/8的磷酸化状态。双基因敲除小鼠的铁负荷比单独缺失Hfe或Tfr2的小鼠更严重；Tfr2基因敲除小鼠的铁负荷比Hfe基因敲除小鼠更大。相对于铁储存，Hfe基因敲除小鼠的铁调素表达降低，Tfr2基因敲除小鼠的值显著更低。在同时缺失Hfe和Tfr2的情况下，铁调素表达进一步降低。基因敲除小鼠肝脏中磷酸化Erk1/2的显著降低以及磷酸化Smad1/5/8的减少表明，丝裂原活化蛋白激酶（MAPK）和骨形态发生蛋白/抗五肢瘫同源物（BMP/SMAD）信号通路可能都参与了Hfe和Tfr2介导的铁调素调节。
这些研究表明，Tfr2缺失导致的铁过载比Hfe缺失更严重，并且两种分子的缺失都会导致明显的铁过载。对Hfe/Tfr2双基因敲除小鼠的分析表明，Hfe和Tfr2通过涉及Erk1/2和Smad1/5/8的平行途径调节铁调素。
