Direct modulation of hepatocyte hepcidin signaling by iron

Liver-secreted hepcidin is the systemic master switch of iron homeostasis and decreased levels of hepcidin are considered to cause iron overload not only in hereditary hemochromatosis but also in hemolytic anemia and chronic liver diseases. The regulation of hepcidin is complex and its response to iron is still not completely understood.

To study the direct effect of iron on various established hepcidin signaling pathways in hepatoma cells or primary hepatocytes.

Hepcidin mRNA expression was studied by quantitative real-time (qRT)-PCR in the presence of various forms of iron including ferric ammonium citrate (FAC) in hepatoma cells (Huh7), murine primary hepatocytes and an established co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and Huh7 cells. To analyze hepcidin signaling, the response to bone morphogenetic protein 6 (BMP6), interleukin (IL)-6, IL-1β, hypoxia and lipopolysaccharide (LPS) were studied. Hepcidin and small mothers against decapentaplegic 6 (SMAD6) mRNA levels were assessed by qRT-PCR and the expression of phosphorylated signal transducer and activator of transcription 3 (phospho-STAT3), STAT3, phospho-SMAD1/5/8 and SMAD1 proteins were analyzed by western blot.

All iron III forms including FAC efficiently blocked hepcidin mRNA expression at non-toxic dosages in Huh7 cells or primary hepatocytes in a time and dose-dependent manner (P < 0.001; P < 0.05). Hepcidin blockage could be efficiently blunted by iron chelators salicylaldehyde isonicotinoyl hydrazone (SIH) and Desferal (P < 0.001). FAC also inhibited BMP6, hypoxia, IL-1β and IL-6-mediated hepcidin induction (P < 0.001; P < 0.001; P < 0.05; P < 0.001), and FAC also inhibited LPS-mediated hepatic hepcidin induction in co-culture model (P < 0.001). Moreover, FAC reduced SMAD6 mRNA and p-SMAD1/5/8 protein expression at basal or upon stimulation by BMP6 (P < 0.05; P < 0.01), and FAC also reduced SMAD6 and p-SMAD1/5/8 expression under hypoxia (P < 0.01; P < 0.05). However, FAC has no significant effect on p-STAT3 protein expression at basal or upon stimulation by various stimuli. Notably, in the presence of the BMP/SMAD signaling pathway inhibitor LDN193189 Hydrochloride (LDN), FAC was unable to further decrease hepcidin, SMAD6 and p-SMAD1/5/8 expression compared with LDN alone.

Iron directly blocks hepatocellular hepcidin signaling through the BMP/SMAD pathway but independent of STAT3. This mechanism may contribute to continued iron overload in many pathophysiological conditions ultimately causing a vicious cycle of continued hepcidin suppression.