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Decreased hepcidin mRNA expression in thalassemic mice

Beta thalassemia major is a congenital haemolytic anaemia resulting from the lack of synthesis of the b-globin chain, a major component of haemoglobin A. In the absence of lifelong transfusions the disease is lethal. When adequate transfusion support is provided the patients suffer from iron overload that can lead to endocrine deficiencies, cirrhosis and cardiac failure that frequently lead to their demise. The iron overload results from ‘transfusional’ iron as well as from an inadequate high intestinal absorption despite high levels of body iron. The understanding of the deranged regulation of intestinal iron absorption is of utmost importance and may lead to rational therapy aimed at decreasing iron overload. Hepcidin, a recently identified anti-microbial peptide expressed in the liver was shown to play a role in conditions associated with both iron overload and iron deficiency. Hepcidin inhibits iron absorption in the proximal small bowel and the release of iron from macrophages. In healthy individuals as well as in murine models hepcidin expression is increased when iron stores are elevated and decreased as a result of iron deficiency anaemia and hypoxia (Nicolas et al, 2001; Park et al, 2001; Pigeon et al, 2001; Ganz, 2003). Thalassemia major represents a unique situation where anaemia, which is expected to decrease hepcidin expression, coexists with iron overload, which ought to increase it. Therefore, it was of interest to study which of these factors has an upper hand in hepcidin expression regulation. We used the recently described severe anaemia model of C57Bl/6 Hbb mice (May et al, 2002) that exhibit anaemia, abnormal red cell morphology, splenomegaly and develop spontaneous hepatic iron deposition (demonstrated by Gomori iron stain of liver tissue), and C57Bl/6 control mice in an attempt to understand the inadequately enhanced absorption of iron in thalassemia. RNA extracted from the liver of adult mice was analysed by quantifiable real time reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the following iron metabolism-related genes: hepcidin, iron-regulated gene 1 (IREG1), neutral gelatinase-associated lipocalin (NGAL), haemochromatosis (HFE), transferrin receptors 1 and 2 (TfR1 and TfR2). The expression levels were normalized to b-actin. Interestingly, despite iron overload the expression levels of both hepcidin and TfR1 were significantly lower (0Æ38-fold) in the thalassemic mice. HFE was moderately decreased (0Æ7-fold), while NGAL was significantly increased (2Æ45-fold). TfR2 and IREG1 did not change significantly (Fig 1). This would suggest that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia. Recently, decreased expression of hepcidin was found in hereditary haemochromatosis in association with elevated levels of nontransferrin bound iron. The elevated expression of NGAL, an alternative iron delivery vehicle, supports the role of nontranferrin bound iron in the abnormal iron regulation in thalassemia. The decreased HFE expression level is similar to the finding in hereditary haemochromatosis (Bridle et al, 2003). The exact mechanism remains to be elucidated. These preliminary findings call for confirmation in human thalassemia patients. If supported, a new therapeutic approach to iron overload states such as thalassemia can be devised based on hepcidin administration or other interventions aimed at overcoming the inadequate response to iron overload associated with elevated levels of non-transferrin bound iron. Fig 1. Expression of iron regulatory genes in the liver of a mouse model of b-thalassemia. cDNA was prepared from the total RNA extracted from the liver of three C57B1/6 Hbb mice and three normal C57B1/6 wild-type mice. Gene expression of six iron regulatory genes was measured using specific primers for quantifiable reverse transcription polymerase chain reaction. Bars represent the average fold change in mRNA expression of each gene in C57B1/6 Hbb mice (black bars), when compared with control mRNA levels in C57B1/6 wild-type mice (grey bars). Each bar represents the average of 2–4 independent experiments, normalized to corresponding b-actin expression. correspondence