A HAMP promoter bioassay system for identifying chemical compounds that modulate hepcidin expression

Hepcidin is the central regulator of systemic iron homeostasis; dysregulation of hepcidin expression causes various iron metabolic disorders, including hereditary hemochromatosis and anemia of inflammation. To identify molecules that modulate hepcidin expression, we developed a bioassay system for hepcidin gene (HAMP) promoter activity by stable transfection of Hep3B hepatoma cells with an expression plasmid in which EGFP was linked to a 2.5-kb human HAMP promoter. Interleukin 6, bone morphogenetic protein 6 (BMP-6), and oncostatin M, well-characterized stimulators of the HAMP promoter, strongly enhanced the green fluorescence intensity of these cells. Dorsomorphin, heparin, and cobalt chloride, known inhibitors of hepcidin expression, significantly suppressed green fluorescence intensity, and these inhibitory effects were more prominent when the cells were stimulated with BMP-6. Employing this system, we screened 1,280 biologically active small molecules and found several candidate inhibitors of hepcidin expression. Apomorphine, benzamil, etoposide, CGS-15943, kenpaullone, and rutaecarpine (all at 10 μmol/L) significantly inhibited hepcidin mRNA expression by Hep3B cells without affecting cell viability. CGS-15943 was the strongest suppressor of BMP-6-induced hepcidin-25 secretion in these cells. We conclude that our newly developed hepcidin promoter bioassay system is useful for identifying and evaluating compounds that modulate hepcidin expression.