VNN1 Regulates The Expression Of Hepcidin In The Liver And The Molecular Mechanism Of Hepatic Iron Disorder

Iron metabolic disorder is one of the most common diseases, which is involved in inflammation, diabetes and cardiovascular disease. Therefore, iron status and its regualtors are important for human health. Hepcidin, a cysteine-rich, iron-inducible peptide secreted by the liver, interacts with ferroportin protein, which localizes on the cell membrane of macrophages and enterocytes, and induces its internalization and degradation. A number of disorders in human, such as hereditary hemochromatosis (HH), anemia of inflammation (AI), chronic kidney diseases and so on, are associated with the deficiency or excess of Hepcidin. However, the regulation of Hepcidin is very complex and depends on many variables, including the status of the systemic iron, inflammation and hypoxia. In particular, Hepcidin levels have been found to be elevated in patients with chronic inflammation, and then resulted in anemia of inflammation (AI). Although it has been indicated that the signal transducer and activator of transcription-3 (STAT-3) signaling mediates the upregulated of Hepcidin during inflammation, the media molecule through which inflammation affects hepcidin expression and iron homeostasis is still not fully understood.VNN1 is a glycosylphosphatidyl inositol (GPI)-anchored pantetheinase highly expressed in gut and liver. It hydrolyzes pantetheine into pantothenic acid (vitamin B5) and cysteamine. The latter is believed to be a key regulator of several essential metabolic pathways, acting through sulfhydryl-disulfide exchange reactions between itself oxidized form, cystamine. In consistance, Vanin-1-/- mice has been shown to be protected from colitis caused by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Since VNN1 controls the production of cysteamine in vivo, the treatment of cystamine completely reversed the protection from colitis in Vanin-1-/-mice. Based on the findings mentioned above, it is easy to confer that there may be a relationship among inflammatory signals, VNN1 and Hepcidin. Therefore, the present study aims to explore the molecular mechanism underlying the regulation of Hepcidin expression and hepatic iron accumulation by VNN1.To answer this question, we used gain- and loss-of-function strategy to manipulate the expression of VNN1 in vitro. We applied adenovirus-mediated VNN1 overexpression and siRNA-mediated interference in mouse primary hepatocytes. Real-time PCR and Western Blot analysis were performed to analyse the expression levels of VNN1 and Hepcidin in the liver. Perls’ Prussian blue staining analysis was carried out to analyse hepatic iron accumulation. At the molecular level, the activation of NF-κB signal pathway was also determined. In addition, luciferase reporter analysis was used to investigate the role of VNN1 in hepcidin transcription regulation.Here we show that lipopolysaccharide (LPS) significantly increased VNN1 and Hepcidin expression levels in mouse primary hepatocytes, which indicates that VNN1 may play a potential role in LPS-induced Hepcidin expression. Overexpression of VNN1 in mouse primary hepatocytes also increased the expression of Hepcidin and hepatic iron accumulation. In contrast, VNN1 knockdown resulted in opposite phenotypes. More importantly, VNN1 knockdown antagonized LPS-induced Hepcidin expression and alleviated iron dysregulation. Our results revealed a positive relationship between VNN1 and Hepcidin expression. Mechanistically, NF-κB signaling pathway plays an important role in inflammation. It is reported that NF-κB binds (-509 bp) in the hepcidin promoter. So we also explored whether NF-κB is involved in the regulation of Hepcidin by VNN1.Our confocal imaging result indicated that VNN1 activates NF-κB nuclear translocation in mouse primary hepatocytes, and BAY 11-7082, an NF-κB antagonist, effectively suppressed this effect. Conversely, VNN1 knockdown antagonized LPS-induced NF-κB activity. These results demonstrate that VNN1 induces hepcidin expression on the transcription level though activating NF-κB pathway. In addition, cystamine, the downstream metabolite of VNN1, completely reversed the inhibition effect of Hepcidin expression by VNN1 knockdown in mouse primary hepatocytes. So it is indicated that at least cystamine partially involved in regulation of Hepcidin expression.In summary, our results strongly suggest that VNN1 induces Hepcidin expression and increases hepatic iron accumulation through activating NF-κB nuclear translocation in mouse primary hepatocytes. In addition, cystamine, the downstream metabolite of VNN1, plays an important role in this regulation. To the best of our knowledge, this is the first time to explore the relationship between VNN1, GPI-anchored paritetheinase, and hepatic iron metabolism, which reveals the versatile biological function of VNN1. According to our results, VNN1 may serve as a pharmaceutical target to treat metabolic diseases.