Direct modulation of hepatocyte hepcidin signaling by iron

ARTICLE HIGHLIGHTS

Research background

Excess iron causes cancer and severe tissue damage and chronic iron overload is not only driving the rather rare hereditary iron overload diseases but also secondary iron overload diseases due to hemolysis or common chronic liver diseases such as alcoholic liver disease or hepatitis C. In most of these diseases, suppression of hepcidin, the systemic master switch of iron homeostasis in mammals, has been identified to play a key role. Hepcidin is primarily expressed in hepatocytes as a precursor pro-peptide and to a lesser extent in macrophages or cardiomyocytes. Elevated hepcidin causes hypoferremia and anemia by efficiently blocking iron absorption, iron recycling and iron storage by binding to and degrading the major iron export pump ferroportin 1.

Research motivation

The direct iron sensing mechanisms by hepcidin remain obscure and seemingly paradox response of hepcidin have been observed in various clinical scenarios. Thus, direct intravenous injection of iron causes rapid induction of hepcidin, iron release in the context of hemolytic diseases such as thalassemia efficiently block hepcidin expression and cause further detrimental iron accumulation. Moreover, it still remains largely unexplained why hepatocellular hepcidin is downregulated under in vitro conditions. These observations prompted us to study in detail the direct effect of iron in cultured hepatocytes.

Research objectives

The authors here aimed to study the direct effect of iron on various established hepcidin signaling pathways including the bone morphogenetic protein (BMP)/small mothers against decapentaplegic (SMAD) signaling pathway and signal transducer and activator of transcription 3 (STAT3)-mediated hepcidin signaling via cytokines, hypoxia, and lipopolysaccharide (LPS) using a recently established macrophage-hepatocyte co-culture model.

Research methods

Hepcidin mRNA expression in presence of various forms of iron was studied, using hepatoma cells (Huh7), murine primary hepatocyte and a co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and hepatoma cells. The response to BMP6, interleukin (IL)-6, IL-1β, hypoxia and LPS were studied in order to analyze hepcidin signaling. Hepcidin and SMAD6 mRNA levels were assessed and the expression of phospho-STAT3, STAT3, phospho-SMAD1/5/8 and SMAD1 proteins were analyzed.

Research results

All iron III forms including ferric ammonium citrate efficiently blocked hepcidin mRNA expression at non-toxic dosages in hepatoma cells or primary hepatocytes. Using iron chelators, the blockage of hepcidin by iron could be efficiently blunted. Iron also had a profound inhibitory effect of basal hepcidin expression and completely abolished BMP6-mediated hepcidin signaling through SMAD but not the STAT3 pathway. Iron also and primarily affected hepcidin even in a typical STAT3-signaling setting through basal modulation of the SMAD pathway and iron significantly attenuated hepcidin response to cytokines, which is SMAD dependent but does not involve STAT3. In the co-culture model, iron inhibited LPS-mediated hepcidin induction.

Research conclusions

In conclusion, iron directly blocks hepatocellular hepcidin transcription involving all forms of iron III and the effect was not caused by toxicity or reduced cell growth. Iron also inhibits hepcidin upregulation in various models of hepcidin stimulation primarily through the BMP/SMAD pathway but independent of STAT3 signaling. We propose that his mechanism may contribute to continued iron overload at least under pathophysiological conditions of iron release ultimately causing a vicious cycle of continued hepcidin suppression and further iron overload.

Research perspectives

This study provides a new concept for better understanding the seemingly paradox response of hepcidin in in vivo and in vitro settings. Moreover, understanding the direct inhibitory effects of iron on hepcidin signaling at the hepatocellular side could help to identify novel molecular targets for future therapies.