Novel hepcidin activators control steady-state hepcidin transcription in a BMP-REs–dependent manner and contribute to the hepcidin responses to IL-6 and BMP-6. (A) The name of each hepcidin promoter reporter construct refers to the elements that have been mutated within the wild-type hepcidin 2.7-kb promoter. B1 and B2 indicate the BMP-RE1 and the BMP-RE2, respectively, whereas S1 indicates the STAT-BS. Reporters driven by the ferroportin or the DAP-kinase promoters were included as specificity controls. (B-C) Shown are the responses of the wild-type and mutated hepcidin promoter to knockdowns of HJV and STAT3 (B), and to knockdowns of the identified hepcidin regulators (C). Results are presented as ratios between the luciferase activity (± SD of Firefly/Renilla) obtained from samples transfected with specific siRNA and samples transfected with control siRNA. Results represent a mean of 5 independent experiments. Statistically significant changes are marked by *(P < .05) or **(P< .005). (D) Novel hepcidin activators are partially involved in the hepcidin responses to IL-6, whereas BMP signals override the requirement for most of the identified modifiers. Huh7 cells were transfected with the indicated siRNAs, starved from serum for 48 hours, and then treated for 15 hours with IL-6 (20 ng/mL) or BMP-6 (10 ng/mL) (supplemental Figure 4). Hepcidin mRNA levels were determined by real-time qPCR analysis. Hepcidin steady-state mRNA level (as shown in Figure 2C) or fold induction of hepcidin mRNA expression in response to stimuli are set to 100% for cells transfected with scrambled siRNA control. The mean of at least 3 independent experiments is shown. Arrows indicate significant changes (P < .05) compared with scrambled siRNA.