Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis.
•Nrf2 regulates HAMP transcription.•Phytoestrogens differentially induce HAMP transcription by Nrf2.•Post-prandial hepcidin induction reduces serum iron and transferrin saturation.•Nrf2 knockout results in iron-overload in mouse embryonic fibroblasts.
ARE, antioxidant response element; Bach1, BTB and CNC homology 1 basic leucine zipper transcription factor 1; BRG1, Brahma-related gene-1; BRM, Brahma; ChIP, chromatin immunoprecipitation; Ct, cycle threshold; c-Jun, Jun proto-oncogene; c-Fos, Finkel-Biskis-Reilly osteogenic sarcoma; DMSO, dimethyl sulfoxide; EMSA, electrophoretic mobility shifts assay; ER, endoplasmic reticulum; FeNTA, Fe(III) nitrilotriacetic acid; IL-6, interleukin 6; LPS, lipopolysaccharide; MT-1, metallothionein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; Hamp, hepcidin antimicrobial peptide; HepcARE, Hepcidin antioxidant response element; HO-1, heme oxygenase 1; IgG, immunoglobulin G; Keap1, Kelch-like ECH-associated protein 1; Maf, musculo-aponeurotic fibrosarcoma; QR1, quinone reductase 1; β-TrCP, β-transducin repeat-containing protein; SCF, Skp, Cullin, F-box; Nrf2, NF-E2-related factor 2; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; tBHQ, tert-butyl hydroquinone; RT-PCR, Real time-PCR; RIPA, Radio-immunoprecipitation assay; Oxidative stress; Iron overload; Hepcidin; Antioxidant response element; Nrf2; Phytoestrogen; Polyphenol; Redox regulation
Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation.
To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression.
Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional.
From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.
Inflammation; Hemojuvelin; TNF-α