Abstract
Production of the iron regulatory peptide hepcidin is tightly controlled by a network of proteins in hepatocytes that sense levels of iron in the circulation (as diferric-transferrin) and in tissues (in ferritin). Human studies show high variability in the normal range of serum hepcidin levels. We have postulated that this may, in part, be related to inter-individual variability in the expression of genes in the iron sensing pathway, potentially governed by epigenetic factors. Here, we have investigated whether genes encoding hepatic iron sensing proteins and hepcidin are regulated by DNA methylation. Experiments were performed on two human hepatoma cell lines, HepG2 cells and Huh7 cells. Basal expression of TFR2 and HAMP was significantly lower in Huh7 cells compared with HepG2 cells. Analysis of bisulphite-converted DNA from Huh7 cells revealed partial methylation of TFR2 (alpha transcript), which could result in gene silencing. Demethylation using 5-aza-2’-deoxycitidine (AZA) increased TFR2 mRNA expression in Huh7. PCR analysis of bisulphite-converted HAMP promoter DNA, using methylation-specific primers, revealed no differences between cell lines. However, HAMP mRNA expression in Huh7 was increased by AZA treatment, suggesting that methylation of one or more iron sensing genes may indirectly influence HAMP expression. Our study provides evidence that DNA methylation might control expression of HAMP and other hepatic iron sensing genes, and indicates that epigenetic influences on iron homeostasis warrant further investigation.
Highlights
Iron homeostasis is maintained by a network of proteins in hepatocytes, which sense changes in circulating and cellular levels of iron
Expression of HAMP and TFR2 alpha was significantly greater in HepG2 cells compared with Huh7 cells (Fig 1A and 1B)
In the context of the differences observed in basal expression of TFR2 alpha and HAMP in the hepatic cell lines, it was interesting to note that there was some variation in the degree of methylation of CpGs in both the TFR2 alpha and HAMP gene promoters in hepatocytes and HepG2 cells (Table 2)
Summary
Iron homeostasis is maintained by a network of proteins in hepatocytes, which sense changes in circulating and cellular levels of iron. Downstream signalling from these sensors leads to the regulated production of hepcidin (reviewed in [1]). Once released from hepatocytes, serum hepcidin acts as a negative regulator of iron export from a number of cell types, including enterocytes and macrophages, by decreasing expression of the iron transport protein ferroportin [2,3,4,5]. In enterocytes, hepcidin limits iron absorption from diet through downregulation of DMT1 [5,6,7,8].
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