Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28–52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein's stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. We conducted a small‐scale chemical screen to identify small molecules that modulate Hepcidin expression. We found that treatment of zebrafish embryos with genistein, a major dietary component of soybeans, enhanced hepcidin transcript levels. By treating human hepatocytes (HepG2), we found that genistein caused a significant increase in Hepcidin transcript levels and promoter activity. We discovered that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the Stat3 binding site or the bone morphogenic protein (BMP) response elements in the Hepcidin promoter. RNA sequencing, chromatinimmunoprecipitation, and ELISA experiments revealed that genistein enhanced BMP and Stat3 signaling in HepG2 cells. In conclusion, genistein is the first nontoxic small molecule identified that increases Hepcidin expression in vivo and in vitro. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. Funding: March of Dimes, NIDDK, US Army Research Office.

4245 Hepcidin, a transcriptionally regulated peptide hormone, produced by hepatocytes in response to iron overload, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes, because its levels are inappropriately low in these patients. Among patients with hereditary hemochromatosis, premenopausal women exhibit less severe iron overload than their male counterparts. While this has been attributed to menstrual blood loss, we hypothesized that estrogens may play a role in regulating Hepcidin expression. We tested estradiol and the phytoestrogens, genistein, apigenin, daizdein, and genistin, for their effects on hepcidin expression in zebrafish embryos. In addition to its estrogenic effects, genistein is known to inhibit multiple protein tyrosine kinases. We found that genistein treatment from 28–52 hours post-fertilization in zebrafish embryos enhanced hepcidin transcript levels relative to liver size, as assessed by whole mount in situ hybridization, while estradiol and the other phytoestrogens did not produce a similar effect. To evaluate whether these effects were conserved in human hepatocytes, we treated HepG2 cells with genistein for 24 hours. We found that genistein treatment was associated with a significant increase in Hepcidin transcript levels, which was abrogated by co-treatment with the bone morphogenic protein (BMP) signaling antagonist dorsomorphin, but not by co-treatment with the estrogen receptor antagonist ICI 182,780. In conclusion, genistein promotes Hepcidin expression in hepatocytes in a BMP-dependent, but estrogen receptor independent manner. Future experiments will address the signaling pathway by which genistein exerts its effect on Hepcidin expression. Disclosures: No relevant conflicts of interest to declare.