Background & aims: Multicopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.
Methods: Mice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped.
Results: Heph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.
Conclusions: These studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.
Keywords: CP, ceruloplasmin; Cp-/-, mice lacking CP in the whole body; DAB, 3,3′-diaminobenzidine; FDR, false discovery rate; FPN1, ferroportin 1; GI, gastrointestinal; HCI, hydrochloric acid; HEPH, hephaestin; Heph-/-, mice lacking HEPH in the whole body; Heph-/-Cp-/- or DKO, double-knockout mice lacking both HEPH and CP; Hephfl/fl, mice with floxed Heph alleles; Hephfl/flCp-/-, mice with floxed Heph alleles and lacking CP in the whole body; Hephint/int, mice lacking HEPH in the intestine alone; Hephint/intCp-/-, mice lacking HEPH in the intestine alone and lacking CP in the whole body; Hephsla/slaCp-/-, mice lacking CP in the whole body and expressing only the sla mutant form of HEPH; Intestinal Iron Absorption; Iron Deficiency Anemia; Iron Overload; MCF, multicopper ferroxidase; NTBI, non-transferrin bound iron; Non-Transferrin Bound Iron; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SD, standard deviation; TBST, Tris-buffered saline with 0.1% Tween-20; TF, transferrin; TIBC, total iron binding capacity; WT, wild-type; sla, sex-linked anemia.