An in vitro hepcidin-FPN AXIS MODEL for exploring the role of macronutrients in cellular iron transport

Abstract Systemic and cellular iron metabolism is tightly regulated, and both its deficiency andexcess lead to major disorders in humans. The liver-derived hormone hepcidin controlssystemic iron balance by binding to ferroportin (FPN), the only known cellular ironexporter. In the duodenum, FPN is expressed on the basolateral surface of intestinalepithelial cells (IECs). Hepcidin-FPN axis is critical for maintaining iron balance, ashepcidin regulates the absorption of dietary iron into the bloodstream by inducing FPNinternalization and degradation in IECs. Disruption of this axis is a key feature of all iron-related disorders. To study cellular iron metabolism in vitro, we developed doxycycline-inducible FPN overexpression in HEK293 and IEC6 cell lines. These cells, based onFPN fused with GFP, were named HEK-FPNGFP and IEC6-FPNGFP, respectively.Strong FPN-GFP expression upon doxycycline treatment and a significant decreasefollowing hepcidin exposure make this an ideal in vitro model with a functional hepcidin-FPN axis. In this study, we used this model in a cell growth-based nutrient deprivationscreen to investigate the roles of major nutrients in cellular iron transport. In our screening, we included glucose titration, reduced the amount of serum in themedia, and depleted essential amino acids. Interestingly, we found that loweringhistidine from the media blocks FPN function. After examining FPN expression, wefound that reducing histidine in the media does not affect the colocalization of hepcidinto the membrane for FPN degradation or alter FPN levels. Although this finding is stillpreliminary, it has been highly reproducible. We are currently trying to understand whathappens to iron levels under reduced histidine media and whether histidine metabolismis necessary for FPN function. This research opens the door to new therapeuticstrategies targeting iron-related disorders, such as anemia and iron overload, byadjusting amino acid availability.