Investigation of the Iron Responsive Elements of the Hepcidin Promoter by In Vivo Imaging of Mice.

Abstract

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. It performs this function by modulating iron transfer by both the intestinal epithelial cells and macrophages. Low or high hepcidin levels result in increased or decreased iron absorption respectively. Hepcidin is mainly liver-derived and regulated at least, in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation have all been shown to influence hepcidin mRNA expression in intact animals. Regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating hepatocytes or mixtures of hepatocytes and macrophages or marrow cells with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains a mystery. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we took advantage of the fact that 2 hepcidin genes exist in mice. Hepcidin 1 is homologous to human hepcidin while hepcidin 2 is the result of a tandem duplication of murine hepcidin 1. In mice hepcidin 1 mRNA expression responds to iron and to inflammation, but hepcidin 2 responds only to iron. We established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals' liver after transfecting hydrodynamically hepcidin promoter/luciferase constructs into mice. Murine hepcidin 1, murine hepcidin 2 and human hepcidin promoters were all shown to have basal luciferase activity. Luciferase expression was lower in HFE knockout mice than in wild type controls. When transfected mice were stimulated with iron or endotoxin, the murine hepcidin 1 and human hepcidin constructs were found to respond to both of these stimuli. The murine hepcidin 2 transfected mice showed a weak response to iron stimulation while there was no stimulation by endotoxin. These findings validate, in an in vivo system, what is known about these promoters from other studies. Subsequent experiments have focused on making shorter hepcidin promoter constructs to identify the key regions of the hepcidin promoter involved in iron induction of hepcidin expression. This strategy will allow us to map the iron response site of the hepcidin promoter and this should aid in identifying transcription factors and upstream stimuli involved in regulating expression of hepcidin by iron.