Graziadei I, Weiss G, Bohm A, Werner-Felmayer G, Vogel W
Department of Internal Medicine, University of Innsbruck, Austria.
J Hepatol. 1997 Oct;27(4):716-25. doi: 10.1016/s0168-8278(97)80089-x.
BACKGROUND/AIMS: We have previously shown that the hepatic acute-phase protein alpha1-antitrypsin (alpha1-AT) is an important mediator of changes in iron metabolism in the course of anaemia of chronic disease. Alpha1-AT profoundly reduces growth of erythroid cells by interfering with transferrin-mediated iron uptake. In the present work we investigate the effects of alpha1-AT on hepatic iron metabolism, as the liver plays a central role in body iron metabolism and in metabolic changes during acute-phase response.
The human hepatoma cell line Hep G2 was cultured in RPMI 1640+10% FCS. The effect of alpha1-AT on transferrin-receptor binding was investigated in equilibrium binding assays with 125I-transferrin. Expression of transferrin receptor was determined by saturation experiments and intracellular ferritin was measured in cell lysates after incubating cells either alone or with alpha1-AT. To determine iron regulatory protein binding activity to iron responsive elements we used gel retardation assays and Northern blot analysis was carried out to investigate transferrin receptor and ferritin mRNA expression.
Alpha1-AT completely prevented transferrin from binding to its receptor and internalization of the transferrin-transferrin receptor complex on HepG2. In addition, alpha1-AT caused a distinct increase in iron regulatory protein binding activity to iron responsive elements, which is characteristic of iron deprivation. Normally, the synthesis of transferrin receptor and ferritin is regulated bidirectionally, but alpha1-AT promoted a unidirectional regulation. Alpha1-AT increased the synthesis of both transferrin receptor and ferritin and, moreover, increased cellular amounts of transferrin receptor mRNA and ferritin H-chain mRNA.
The effect of alpha1-AT on transferrin receptor synthesis appears to be mediated via activation of iron responsive element binding affinity of iron regulatory protein leading to an increased stability of transferrin receptor mRNA. Changes in ferritin, however, may be related to a transcriptionally mediated, iron-independent pathway which overrides the influence of activated iron regulatory protein. These specific changes in iron metabolism are the very ones seen in the course of anaemia of chronic disease. Our results emphasize the central role of alpha1-AT as a mediator of altered iron metabolism, characteristic of anaemia of chronic disease, not only with respect to erythroid cells but also with respect to liver cells.
背景/目的：我们之前已经表明，肝脏急性期蛋白α1-抗胰蛋白酶（α1-AT）是慢性病贫血过程中铁代谢变化的重要介质。α1-AT通过干扰转铁蛋白介导的铁摄取，显著降低红系细胞的生长。在本研究中，我们研究了α1-AT对肝脏铁代谢的影响，因为肝脏在机体铁代谢以及急性期反应期间的代谢变化中起着核心作用。
人肝癌细胞系Hep G2在RPMI 1640 + 10%胎牛血清中培养。用125I-转铁蛋白通过平衡结合试验研究α1-AT对转铁蛋白受体结合的影响。通过饱和实验测定转铁蛋白受体的表达，并在细胞单独培养或与α1-AT共同培养后，在细胞裂解物中测量细胞内铁蛋白。为了确定铁调节蛋白与铁反应元件的结合活性，我们使用凝胶阻滞试验，并进行Northern印迹分析以研究转铁蛋白受体和铁蛋白mRNA的表达。
α1-AT完全阻止转铁蛋白与其受体结合以及转铁蛋白-转铁蛋白受体复合物在HepG2细胞上的内化。此外，α1-AT导致铁调节蛋白与铁反应元件的结合活性显著增加，这是铁缺乏的特征。正常情况下，转铁蛋白受体和铁蛋白的合成是双向调节的，但α1-AT促进了单向调节。α1-AT增加了转铁蛋白受体和铁蛋白两者的合成，而且增加了转铁蛋白受体mRNA和铁蛋白H链mRNA的细胞含量。
α1-AT对转铁蛋白受体合成的影响似乎是通过激活铁调节蛋白与铁反应元件的结合亲和力介导的，从而导致转铁蛋白受体mRNA稳定性增加。然而，铁蛋白的变化可能与转录介导的、不依赖铁的途径有关，该途径超越了激活的铁调节蛋白的影响。这些铁代谢的特定变化正是在慢性病贫血过程中所见到的。我们的结果强调了α1-AT作为铁代谢改变的介质的核心作用，这种改变是慢性病贫血的特征，不仅涉及红系细胞，也涉及肝细胞。
