SMAD7 antagonizes SMAD4-mediated responses. (A) The name of each construct refers to the elements that have been mutated compared with the WT_2.7kb construct. B1 and B2 indicate BMP-RE1 and BMP-RE2, respectively, and S1 and S2 indicate SMAD-RE1 and SMAD-RE2, respectively. (B) Steady-state hepcidin promoter activity was measured from the SMAD_RE1_2.7kb luciferase reporter construct, compared with WT_2.7kb promoter, and is presented as a fold change ± SD of Firefly/Renilla ratios. (C-D) Huh7 cells were transfected with siRNAs directed against SMAD7 (C), SMAD4 (D), or scrambled siRNA as a control. Forty-eight hours later, cells were transfected with luciferase reporter vectors. Luciferase activity was measured 24 hours later. 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 4 independent experiments. Statistically significant changes between the WT promoter and the mutated promoter (C) or between the SMAD4 siRNA transfection and scrambled controls (C) are marked by an asterisk (*P < .05, **P < .005, and ***P < .001). (E) SMAD7, a potent hepcidin suppressor, counteracts the BMP/SMAD-mediated induction of hepcidin expression. SMAD7 is coregulated with hepcidin via SMAD4-dependent signaling pathways and thus antagonizes SMAD4-mediated responses of the hepcidin promoter. SMAD7 expression is further modulated by cytokines independent of SMAD4 signaling.