Hypoxia Enhances H2O2-Mediated Upreguation of Hepcidin: Potential Role of Oxidases in Iron Regulation

The liver-secreted peptide hepcidin plays a major role in the regulation of iron homeostasis being also critical for the pathological liver iron accumulation in alcoholic liver disease (ALD). Iron, inflammation, cytokines or H₂O₂ are able to upregulate hepcidin, however the role of hypoxia in combination with low levels of H₂O₂ is poorly understood. We here study hepcidin signaling in liver cells under hypoxia and H₂O₂ and the involvement of oxidases, such as NOX4, as potential upstream hepcidin regulators.

Two hypoxic systems (hypoxia chamber and the enzymatic GOX/CAT system) were used to maintain low oxygen level (5% O₂) over 24 hours in Huh7 hepatoma cells and in THP-1 macrophages, the latter also allowing to independently control low steady state (ss) H₂O₂ levels. Hepcidin and NOXs mRNA levels were studied by qRT-PCR. Cellular hypoxia was confirmed by staining of pimonidazole adducts and H₂O₂ levels were analyzed by Prx2 western blot. The involvement of the liver expressed NADPH oxidase 4 (NOX4) as well as urate oxidase (UOX) was studied by overexpressing of NOX4 or UOX promoter constructs. In addition, we used THP-1 macrophages to study the role of NOX2, predominantly expressed in Kupffer cells, in regulating hepcidin.

We first show that hypoxia generated either by hypoxia chamber or the GOX/CAT system induced hepcidin mRNA in Huh7 cells over 24h. Hypoxia-mediated induction of hepcidin was further potentiated during co-exposure of cells with low ss H₂O₂ levels predominantly at the transcriptional level via STAT3 signaling pathway. Both, Prx2 oxidation and STAT3 phosphorylation mirrored intracellular H₂O₂ and changes in hepcidin mRNA levels. Notably, NOX4 was also strongly upregulated in hepatocytes during hypoxia, suggesting a mechanistic role in mediating hepcidin upregulation. Overexpression of NOX4 increased hepcidin mRNA and this effect was even higher at low O₂ levels. Moreover, similar results were obtained after overexpression of UOX in Huh7 cells. Our findings could be finally validated in THP-1 macrophages, which also express both, NOX2 and hepcidin. In THP-1 cells, the activation of protein kinase C signaling by treatment with PMA led to a strong NOX2 and hepcidin mRNA induction. In contrast, under hypoxia NOX2 and hepcidin mRNA levels were maintained.

We here show that hypoxia drastically enhances the H₂O₂-mediated induction of hepcidin via STAT3 in hepatocytes and found evidence for an oxidase, such as NOX4, as efficient upstream regulator of hepcidin. In macrophages, NOX2 has shown also to be capable of regulate hepcidin, although, contrary to NOX4, it is not responsive to hypoxia.