Novel Ferroxidases in Mammalian Intestinal Epithelial Cells

Abstract

Hephaestin (HEPH) is an intestinal ferroxidase (FOX) that has been implicated as essential for intestinal iron absorption. HEPH oxidizes ferrous iron exported by ferroportin 1 (FPN1), to allow binding of ferric iron to transferrin in the blood. We have previously discovered: 1) the existence of a soluble, intracellular form of HEPH, and 2) that additional novel FOXs in mammalian enterocytes. The purpose of the current study was to test the hypothesis that novel FOXs exist in enterocytes that compensate for the lack of HEPH. A HEPH knockdown (KD) Caco-2 cell line was developed by stable transfection of HEPH-specific or negative control, plasmid-derived shRNAs. 59Fe flux was measured in fully-differentiated Caco-2 cells grown on collagen-coated, bicameral cell culture inserts. HEPH KD enhanced iron uptake (2-fold) under control conditions and enhanced iron efflux (>6-fold) under iron-deficient conditions. Increased FPN1 expression in HEPH KD cells may provide a mechanistic explanation for enhanced iron efflux. FOX activity was then assessed using a spectrophotometric transferrin-coupled assay. Consistent with enhanced iron efflux, HEPH KD and iron deprivation increased cytosolic and membrane FOX activity. Next, intestine-specific HEPH knockout (KO) and wild type (WT) male and female mice were fed control or iron-deficient diets for 5-6 weeks. Intestinal iron absorption was subsequently tested by oral gavage of 59Fe. Adult HEPH KO mice had normal hemoglobin levels and absorbed iron as efficiently as WT littermates. In conclusion, HEPH is not required for efficient iron transport and novel FOXs exist in mammalian enterocytes that compensate for the lack of HEPH.