Direct modulation of hepatocyte hepcidin signaling by iron

Figure 2 Ferric ammonium citrate profoundly blocks bone morphogenetic protein 6-mediated hepcidin signaling. Huh7 cells were treated with or without bone morphogenetic protein 6 (BMP6) (40 ng/mL) in the presence or absence of ferric ammonium citrate (FAC) (50 μmol/L) for 24 h. Total RNA and protein were extracted from Huh7 cells. A: FAC decreased the hepcidin mRNA expression in the presence or absence of BMP6; B: FAC decreased small mothers against decapentaplegic 6 (SMAD6) mRNA expression in the presence or absence of BMP6; C, D: FAC decreased p-SMAD1/5/8 protein expression in the presence or absence of BMP6; E, F: Both BMP6 and FAC have no significant effect on phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein expression. SMAD1, p-SMAD1/5/8, STAT3, p-STAT3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels were determined by Western blotting. Hepcidin and SMAD6 mRNA levels were determined by qRT-PCR, normalized to GAPDH. Western Blots are representatives of three independent experiments. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs control; ^dP < 0.01, ^eP < 0.001 vs BMP6 group. FAC: Ferric ammonium citrate; BMP6. Bone morphogenetic protein 6; p-: Phospho-; SMAD: Small mothers against decapentaplegic; STAT3: Signal transducer and activator of transcription 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.