Direct modulation of hepatocyte hepcidin signaling by iron

Figure 3 Ferric ammonium citrate efficiently inhibits hypoxia-mediated hepcidin response independent of signal transducer and activator of transcription 3. Huh7 cells were exposed to normoxia (210 mL/L O₂, 21% O₂) or hypoxia (10 mL/L O₂, 1% O₂) 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 basal and hypoxia-induced hepcidin mRNA expression; B: Hypoxia has no obvious effect on small mothers against decapentaplegic 6 (SMAD6) mRNA expression, but FAC decreased SMAD6 mRNA expression in the presence or absence of hypoxia; C, D: Hypoxia has no significant effect on p-SMAD1/5/8 protein expression, while FAC decreased p-SMAD1/5/8 protein expression in the presence or absence of hypoxia; E, F: Hypoxia increased phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein expression, while FAC has no significant effect on p-STAT3 protein expression in the presence or absence of hypoxia. 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 (21% O₂); ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs control (1% O₂). FAC: Ferric ammonium citrate; O₂: oxygen; p-: Phospho-; SMAD: Small mothers against decapentaplegic; STAT3: Signal transducer and activator of transcription 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.