Hepcidin is a transcriptionally regulated peptide hormone that is expressed primarily in the liver and excreted in urine. It is up-regulated in response to inflammation[2, 3] or iron overload and down-regulated in response to increased erythropoiesis, iron deficiency, or hypoxia. Hepcidin decreases intestinal iron absorption and macrophage iron release by causing internalization of the iron exporter, ferroportin1.[5-7] Patients with hereditary hemochromatosis or thalassemia[9-11] exhibit inappropriately low levels of hepcidin and increased intestinal iron absorption, despite the presence of systemic iron overload. Although treatment for iron overload is currently based on removal of blood or administration of iron chelators, it may be possible to prevent iron overload in patients with genetic predisposition if nontoxic small molecules can be administered that increase transcription of Hepcidin.
Iron overload and inflammation[2, 3] stimulate hepcidin expression by triggering the mothers against decapentaplegic homolog (Smad)-signaling or signal transducer and activator of transcription (Stat)-signaling pathways, respectively. It has been demonstrated that exposing human hepatocytes to bone morphogenic proteins (BMPs) up-regulates Hepcidin transcription by increasing Smad4 binding at Smad4-binding motifs, termed BMP response elements (BREs), in the Hepcidin promoter.[13-16] BMPs are members of the transforming growth factor beta (TGF-β) family that signal by binding to transmembrane receptor complexes with serine-threonine kinase activity. Recent studies in mouse models[18-21] indicate that BMP6 is the most likely physiologic regulator of hepcidin transcription in response to iron loading. Inflammatory stimuli, on the other hand, trigger increased serum interleukin-6 (IL-6) levels. IL-6 stimulates Hepcidin expression through increased Stat3 binding to a Stat3-responsive element in the Hepcidin promoter.[24-27]
We have developed the zebrafish embryo (Danio rerio) as an in vivo model to study hepcidin expression. Hepcidin expression begins at 36 hours postfertilization (hpf) in the zebrafish embryo and is responsive to iron levels and BMPs during embryonic development. To demonstrate that zebrafish embryos can be used to identify small-molecule modulators of Hepcidin expression, we screened a small number of naturally occurring isoflavones and related molecules for their effect on Hepcidin expression. We chose to evaluate isoflavones because they are nontoxic and are known to have kinase inhibitory actions. In this way, we identified genistein as the first small-molecule experimental drug to increase Hepcidin expression in vivo. We found that genistein also increased Hepcidin expression in cultured human hepatocytes (HepG2 cells). Using luciferase reporter assays, RNA sequencing (RNA-seq), and chromatin immunoprecipitation (ChIP), we demonstrated that genistein increases Hepcidin expression in a Smad4-dependent and Stat3-dependent manner.