NCOA4 Expression in Hepatic Cells Is Upregulated Under Physiological and Pathophysiological Conditions Associated with Hypoxia

The ubiquitously expressed intracellular protein NCOA4 mediates the degradation of ferritin in vitro (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014) and its loss disrupts systemic iron balance in mice (Bellelli et al., Cell Rep 2016). While a mechanism for increased NCOA4 protein turnover under high iron conditions has been reported in transformed cell lines of non-hepatic origin (Mancias et al., Elife, 2015), factors that regulate NCOA4 mRNA levels have not yet been described. Here we investigate stimuli that induce NCOA4 mRNA expression in hepatoma cells as well as in liver, the major iron depot of the body.

We found that treating the human hepatoma cell line, Hep3B, for 18 hrs with increasing concentrations of the iron chelator desferrioxamine (DFO; 25-100μM) resulted in significantly higher NCOA4 mRNA (ANOVA P<.0001; 1.8 fold increase at the highest dose). Additionally, the murine hepatoma cell line, Hepa1-6, responded to 18 hrs of DFO treatment (25-100μM) with a dose-dependent rise in Ncoa4 mRNA levels (ANOVA P<.0001; 2.7 fold increase at the highest dose). DFO treatment of Hep3B and Hepa1-6 cells also increased mRNA levels of the transferrin receptor, confirming induction of iron deficiency. Because DFO has also been shown to stabilize hypoxia inducible factor (HIF), we examined the effects of other chemical hypoxia mimetics on hepatoma cell lines. Hep3B cells treated for 18 hrs with CoCl₂ (25-75 μM), which is known to enhance the stability of HIF1α, showed a 2-fold increase in NCOA4 mRNA (ANOVA; P<.0001). Similarly, treatment of Hep3B cells with dimethyloxalylglycine (DMOG; 1mM), a competitive inhibitor of HIF prolyl-hydroxylases that promote HIF-α degradation, resulted in a 3-fold increase in NCOA4 mRNA at 12 hrs, which was further enhanced at 18 hrs. These results suggest that hepatoma cells in culture may respond to decreased iron availability and/or hypoxia by upregulating NCOA4 mRNA levels.

We also investigated if Ncoa4 mRNA expression is upregulated in mouse livers under conditions associated with depletion of hepatic iron stores and/or hypoxia. C57BL/6N mice raised on a 45 p.p.m. iron diet were subjected to a large-volume phlebotomy (500μl; accompanied by intraperitoneal saline volume replacement) and fed an iron-deficient diet (2-6 p.p.m.) after bleeding. Seven days after phlebotomy, mice showed significantly lower blood hemoglobin levels (Student's T test P<.0001) and hepatic non-heme iron concentrations (P<.0001). However, the phlebotomized mice exhibited 1.8-fold higher hepatic Ncoa4 mRNA levels (P<.01) compared to non-phlebotomized controls. Additionally, we examined if hepatic Ncoa4 expression changed during normal pregnancy, a state in which extra-hepatic iron demands are increased by both the expanding maternal blood volume and the growing fetus. Compared to non-pregnant females, C57BL/6N mice at day 18.5 of pregnancy showed significantly lower liver non-heme iron concentrations (P<.01), while hepatic Ncoa4 mRNA levels were 3.5-fold higher (P<.01) in pregnant females compared to non-pregnant controls. By mining a published murine gene expression dataset (GEO accession GDS5818), we found that hepatic Ncoa4 mRNA levels are relatively high in early life (embryonic day 18 and postnatal day 5) and decline by adulthood (postnatal day 56), suggesting a relatively greater hepatic requirement for NCOA4 function during periods of high growth. Collectively, these in vivo data show that Ncoa4 upregulation correlates with conditions in which mobilizing iron for systemic use takes precedence over building hepatic iron stores.

In summary, we show for the first time that Ncoa4 mRNA rises significantly in physiological and pathophysiological states in which increased extra-hepatic iron demands induce a reduction in hepatic iron stores. Additionally, we show that Ncoa4 mRNA levels in cultured cells of hepatic origin rise in response to hypoxia mimetics. Because NCOA4 functions as a cargo receptor that traffics ferritin to the autolysosome, NCOA4 protein is expected to be consumed during the process of ferritinophagy. We suggest that upregulation of hepatic Ncoa4 mRNA in response to hypoxia may represent a physiological adaptation to replenish NCOA4 protein that is needed to support continued mobilization of iron from the liver during periods of increased systemic iron demands.