Global Transcriptional Response to Hfe Deficiency and Dietary Iron Overload in Mouse Liver and Duodenum
Alejandra Rodriguez1*, Tiina Luukkaala2, Robert E. Fleming3,4,5, Robert S. Britton5,6, Bruce R. Bacon5,6, Seppo Parkkila1,7

https://doi.org/10.1371/journal.pone.0007212

Summary

This study  was based on microarray analysis to study the comparison of gene expression of wild-type mice fed a regular diet to that of Hfe2/2 mice and carbonyl iron-loaded mice. The entire transcriptome can be profiled for expression using current RNA microarray technologies. This approach has been utilized to investigate how the liver and duodenum, two organs with significant roles in iron metabolism, respond to the disruption of the Hfe gene on mRNA expression. The identification of genes whose expression is altered by iron excess and those, whose expression is differently influenced by a lack of Hfe protein was made possible by this method. Similarly, effects of Hfe deficit and dietary iron overload were seen in 11 upregulated and 7 downregulated genes, while these two circumstances differentially regulated 27 genes in the liver. Using both microarray analysis and Q-RT-PCR, the expression of Hamp1, Hamp2, Lcn2, and Cp was increased as shown in (Figure 1). 24 different genes were represented by a total of 29 results from the hepatic microarray data that were examined by Q-RT-PCR; 27 (93.1%) of these findings demonstrated agreement between the two techniques. In mice given an iron-supplemented diet, Hmox1 expression was increased while Tfrc expression was downregulated in the duodenum validated with Q-RT-PCR. Distinct 17 genes mRNA expression levels were examined, and 16 (94.1%) of them had results, that were consistent with microarray analysis and Q-RT-PCR shown in (Figure 2 & 3). Functional annotation of the list of regulated genes reveals an enrichment of cytochrome P450 proteins as well as other proteins involved in glutathione metabolism, acute-phase response, organic acid biosynthesis, and cellular iron homeostasis, among other processes. This research showed that heat shock proteins and proteins involved in inflammatory reactions or antigen processing were enriched. Endopeptidase activity, lipid catabolism, and antimicrobial activity were among the functional categories that were overrepresented among the genes controlled by Hfe deficiency. According to these findings, deletion of the Hfe gene causes the production of numerous genes encoding for digestive enzymes, including elastases, carboxypeptidases, trypsins, chymotrypsins, amylases, and lipases, in the duodenum.

Figure 1. Expression of genes affected by dietary iron overload in the liver, as confirmed by Q-RT-PCR. Samples from 5 mice fed an iron-supplemented diet and 4 mice fed a control diet were used, and each sample was run in triplicate. (mean6SD). *p,0.05; **p,0.025; ***p,0.01.

Figure 2. Expression of genes regulated in the duodenum of dietary iron-loaded mice as verified by Q-RT-PCR. Samples from 5 mice fed an iron-supplemented diet and 4 mice fed a control diet were used, and each sample was run in triplicate. (mean6SD). *p,0.05;**p,0.025;***p,0.01.

Figure 3. Q-RT-PCR validation of the duodenal microarray data from Hfe2/2 mice. Samples from 4 mice were used for the Hfe2/2 and control groups, and each sample was examined three times. (mean6SD). *p,0.05; **p,0.025; ***p,0.01.