Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors

Christina Warnecke,Ulrike Maegdefrau,Daniel P Gale,Gunnar Schley,Melanie Volke,Bernd Klanke,Carmine Zoccali,Kai-Uwe Eckardt,Anja-Katrin Bosserhoff,Patrick H Maxwell

doi:10.1371/journal.pone.0007875

Abstract

BackgroundHepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.Methodology/Principal FindingsHepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.Conclusions/SignificanceTaken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.

Highlights

The iron regulatory peptide hepcidin is a liver-derived acute phase peptide and a key regulator of systemic iron metabolism [1,2,3,4]
Much information about the determinants controlling hepcidin expression was obtained from the genetics of hereditary hemochromatosis [12], which is characterized by insufficient hepcidin levels due to mutations in the transferrin receptor 2 (TfR2) gene, the hemochromatosis genes HFE and HFE2, or the hepcidin gene itself
We first analyzed the response of hepcidin mRNA levels to hypoxia and chemical hypoxia-inducible transcription factor (HIF) stabilization in HepG2 cells

Summary

Introduction

The iron regulatory peptide hepcidin (gene name : HAMP) is a liver-derived acute phase peptide and a key regulator of systemic iron metabolism [1,2,3,4]. Interleukin-6 (IL-6)-stimulated hepcidin induction is mediated by a highly conserved STAT3 binding element in the proximal promoter of the HAMP gene [9,10]. This sequence motif controls both IL-6-induced, as well as basal, HAMP promoter activity. Liver-specific SMAD4 (the common downstream mediator for all TGF-b superfamily ligands) knock-out mice exhibit marked iron accumulation and fail to increase hepcidin expression in response to TGF-b1, BMP-4, IL-6 or iron overload, suggesting a common role for SMAD4 in the manifold pathways of hepcidin regulation [14]. Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. We investigated the role of HIF in hepcidin regulation

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