Global Transcriptional Response to Hfe Deficiency and Dietary Iron Overload in Mouse Liver and Duodenum

Introduction

Iron plays crucial roles in cellular metabolism but, in excess, it can catalyze the formation of free radicals leading to oxidative stress and cell damage [1]. Iron is absorbed in the duodenum, where it crosses the apical and basolateral membranes of absorptive enterocytes to enter the blood stream [2]. There is no regulated mechanism of iron excretion, and thus the absorption of iron must be tightly regulated to maintain iron balance. HFE-related hereditary hemochromatosis (HH, OMIM-235200) is an autosomal recessive disorder in which absorption of iron is inappropriately high [3], [4]. HH is characterized by high transferrin saturation and low iron content in macrophages. Iron is deposited primarily in the parenchymal cells of various organs, particularly the liver, but also the pancreas, heart, skin, and testes, resulting in tissue damage and organ failure. Clinical complications in untreated HH patients include hepatic fibrosis, cirrhosis, hepatocellular carcinoma, diabetes, cardiomyopathy, hypogonadism, and arthritis [4].

HH is characterized by inappropriately low expression of the iron-regulatory hormone hepcidin [5]. Hepcidin, a small peptide hormone expressed mainly in the liver, is a central player in the maintenance of iron balance [6]. The only known molecule capable of transporting iron out of cells is ferroportin [7]–[9]. This iron exporter is located in the plasma membrane of enterocytes, reticuloendothelial cells, hepatocytes, and placental cells [7]. Hepcidin binds to ferroportin and induces its internalization and degradation, therefore suppressing the transport of iron into the circulation [10]. The expression of hepcidin is induced by increased iron stores and inflammation, and suppressed by hypoxia and anemia [11], [12].

Mice homozygous for a null allele of Hfe (Hfe^−/−) provide a genetic animal model of HH [13]. There are several animal models of iron overload based on administration of exogenous iron [14]. According to the route of iron delivery, these can be divided into two main types: enteral (i.e. dietary) and parenteral models. For example, dietary supplementation with carbonyl iron in mice reproduces the HH pattern of hepatic iron loading, with predominantly parenchymal iron deposition [14]. Although both Hfe ^−/− mice and carbonyl iron-fed mice develop iron overload, there are important differences between these two models. Hfe ^−/− mice lack Hfe protein and therefore have decreased expression of hepcidin [15], [16], while mice with dietary iron overload express functional Hfe protein and their hepcidin expression is elevated [12].

Current RNA microarray technology allows expression profiling of the whole transcriptome. This methodology has been used to explore the effects of Hfe gene disruption on mRNA expression in the liver and duodenum, two organs with crucial roles in iron metabolism [17]. In the present study, we used this approach to study gene expression in the liver and duodenum of Hfe ^−/− mice and wild-type mice, with or without dietary iron overload. This allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein.

Results

We used global microarray analysis to study gene expression in the liver and duodenum of Hfe ^−/− mice and carbonyl iron loaded mice, and comparing it with that of wild-type mice fed a standard diet. This approach allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein. All the mice used were males and all had the same genetic background (C57BL/6).

Discussion

The goal of this study was to explore and compare the genome-wide transcriptome response to Hfe deficiency and dietary iron overload in murine liver and duodenum. This approach allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein. The global transcriptional response to Hfe deficiency has been explored previously in the liver and duodenum of two mouse strains [17]. However, it is notable that only a few analogous changes in gene expression are seen when comparing our data with those of the previous study, even for mice of the same genetic background. Two other reports have explored expression of selected genes by using dedicated arrays in Hfe^−/− mice and in mice with secondary iron overload produced by intraperitoneal injection of iron-dextran [19], [20]. In one study, duodenum and liver samples were analyzed using an array of iron-related genes [19]. The results for duodenal gene expression in Hfe^−/− mice have no concordance with ours. Regulation of hepatic gene expression, on the other hand, is similar for several genes, such as Hamp1, Tfrc and Mt1. The second report focused on gene expression in the duodenum [20], and again, there is little concordance between their observations and ours. The lack of agreement between these studies is probably due to differences in the animal models (parenteral vs. enteral iron loading; mouse strains) and in the microarray methodology.

The hepatic expression of acute phase proteins (APPs) can be induced by inflammatory mediators such as interleukin-6. Interestingly, the liver of Hfe^−/− mice has upregulated expression of APPs such as serum amyloids, lipocalins and orosomucoids. Notably, the expression of serum amyloid genes (Saa1, Saa2, Saa3) was upregulated in the Hfe^−/− mice compared to being downregulated in dietary iron overload, suggesting that Hfe deficiency induces this gene expression by an iron-independent mechanism. However, hepatic interleukin-6 mRNA expression was not significantly changed by Hfe deficiency, so the potential involvement of this cytokine in the observed upregulation of APPs remains uncertain.

Lipocalin2 (human Ngal from neutrophil gelatinase-associated lipocalin) is an APP with antimicrobial properties through a mechanism of iron deprivation by siderophore binding [21]. It can donate iron to various types of cells [22], [23] and seems to be capable of intracellular iron chelation and iron excretion [24]. Furthermore, a recent study has shown that lipocalin2 is an adipokine with potential importance in insulin resistance associated with obesity [25]. We observed that Lcn2 expression is increased in the liver of both Hfe^−/− mice and those with dietary iron overload, suggesting that this induction is iron-related.

Dietary iron overload of the liver led to increased expression of both hepcidin genes (Hamp1, Hamp2) as previously reported [26], [27], and these results were verified by Q-RT-PCR. In the liver of Hfe ^−/− mice, Hamp1 expression was downregulated as expected [15], [16], [19]. We also examined the levels of Hamp2 mRNA by Q-RT-PCR and found a -1.92-fold change. The low expression of hepatic Hamp1 in Hfe ^−/− mice is likely responsible for the increased iron absorption and low microphage iron content in these mice [15], [16], [19].

Inhibitor of DNA-binding/differentiation proteins, also known as Id proteins, comprise a family of proteins that heterodimerize with basic-helix-loop-helix (bHLH) transcription factors to inhibit their binding to DNA. Several studies have reported that Id proteins have important roles in differentiation, cell cycle and angiogenesis in various cell types [28]. Expression of Id1, 2, and 3 is increased during liver disease, with levels that escalate as liver disease progresses from hepatitis to cirrhosis. In hepatocellular carcinoma, high expression is observed in well-differentiated tumors, and it decreases as the tumor cells become undifferentiated [29]. In light of these findings, it has been suggested that Id1, 2, and 3 may play a role in the early stages of hepatocarcinogenesis. Given that, it is notable that we found that the expression of Id1, 2, 3, and 4 was increased in the liver of mice with dietary iron overload, but was unaffected in Hfe ^−/− mice. Increased hepatic expression of Id1 mRNA has previously been reported in mice fed an iron-supplemented diet [30]. The same study showed upregulation of the gene for bone morphogenetic protein 6 (Bmp6) in the same experimental mice. Recent work demonstrates that Bmp6 is a key player in the signalling pathway that controls hepcidin expression [31]. Unexpectedly, upregulation of hepatic Bmp6 mRNA expression by dietary iron overload was not evident in the current study.

The gene expression of several heat shock proteins was downregulated in the liver and duodenum by both Hfe deficiency and dietary iron overload, with a considerably greater number of these genes downregulated in the liver of Hfe−/− mice. Although these genes are induced under certain stress conditions, such as heat shock and ischemia–reperfusion, their expression is decreased by iron overload [19], [27], [32]. Currently, the physiological implications of this downregulation are unknown.

Our results indicate that disruption of the Hfe gene induces the expression of many genes in the duodenum coding for digestive enzymes, such as elastases, carboxypeptidases, trypsins, chymotrypsins, amylases, and lipases. In contrast, feeding mice with an iron-supplemented diet did not affect the expression of any of these genes. The upregulation of gene expression for digestive enzymes in Hfe ^−/− mice is surprising because overexpression of these enzymes has not been associated with HH.

A common feature of the duodenal response to both Hfe deficiency and dietary iron overload was the transcriptional repression of genes involved in antimicrobial activities, such as cryptdins. In mice fed an iron-supplemented diet, there was also a decrease in mRNA expression for genes involved in antigen processing and presentation, such as some genes of the MHC class II family.

The solute carrier molecules constitute a large family of proteins involved in membrane transport of diverse molecules. The gene expression of many family members was affected by Hfe deficiency or dietary iron overload. In the duodenum, the expression of the sodium-coupled neutral amino acid transporter Slc38a5 was induced in Hfe ^−/− mice and repressed in mice fed an iron-supplemented diet. In the liver, the expression of Slc46a3 was upregulated in Hfe ^−/− mice. This gene belongs to the Slc46 subfamily of heme transporters. It is thus a close relative of Slc46a1 (also known as HCP1), a recently identified, although controversial, heme transporter [33], [34]. The iron transporter Dmt1, encoded by Slc11a2, contains an iron-responsive element (IRE) in the 3′UTR of its mRNA. This permits the regulation of Dmt1 mRNA levels according to the cellular labile iron pool by mediation of the iron regulatory proteins, IRP1 and IRP2. Under iron-replete conditions, IRP activity is reduced rendering the Dmt1 mRNA vulnerable to degradation. The opposite is true under iron-deficient conditions, which is believed to be the situation inside the enterocytes of HH patients and Hfe ^−/− mice [35], [36]. Accordingly, in some studies, increased expression of Dmt1 has been observed in the duodenum of HH patients [37] as well as in Hfe ^−/− mice [38]. However, we did not find a significant change in the expression of Dmt1 in the Hfe ^−/− duodenum. This may be explained by the inability of our microarray probes and PCR primers to discriminate between IRE-positive and IRE–negative transcripts.

The post-transcriptional regulation of Tfrc (transferrin receptor 1) by iron is also mediated through the IRE/IRP system [39]. Tfrc is involved in the uptake of transferrin-bound iron by cells. Analogous to our observations, suppression of Tfrc expression in the duodenum [40] and liver [27] of mice fed an iron-supplemented diet, and in the liver of Hfe^−/− mice [19] has been reported previously. Our microarray analysis indicates that the expression of Tfrc was not significantly changed in the duodenum of Hfe ^−/− mice, a result that agrees with a previous report [19].

Excess free iron increases oxidant production [1]. Subsequently, some antioxidant defense mechanisms are upregulated in order to provide resistance to iron-related toxicity. It is notable from our data that this response is elicited in both liver and duodenum, as seen in the upregulation of glutathione S-transferase genes. Interestingly, dietary iron overload seems to induce a stronger response than Hfe deficiency, especially in the regulation of enzymes involved in glutathione-related detoxification of reactive intermediates.

In conclusion, Hfe deficiency results in increased gene expression of hepatic APPs and duodenal digestive enzymes. In contrast, dietary iron overload causes a more pronounced change of gene expression responsive to oxidative stress.

Materials and Methods

Supporting Information