Development and characterization of Hephaestin knockdown Caco‐2 cells (818.13)

Abstract

Intestinal transport of inorganic iron has been intensively studied. Ferric iron is reduced, possibly by duodenal cytochrome b (Dcytb), and transported into cells by divalent metal‐ion transport 1 (Dmt1). Ferrous iron is exported from enterocytes via ferroportin 1 (Fpn1) and is then oxidized prior to binding to transferrin. The efflux step, involving the export‐oxidation functional couple, is the rate‐limiting step in iron absorption. Although an iron oxidase has been discovered (Heph), it is likely that other oxidases also exist. The current study was thus designed to assess the necessity of Heph for iron transport using the Caco‐2 cell model. Caco‐2 cells, which differentiate in post‐confluent culture, express an inducible, vectorial iron transport system, faithfully recapitulating mammalian iron absorption. Heph was effectively silenced in Caco‐2 cells using shRNA technology. A control cell line was also established with negative‐control shRNA transfected cells. Heph KD cells grew normally and were visually indistinguishable from control cells. Gene expression studies showed that Dcytb (~55%) and Dmt1 (~25%) mRNA expression was decreased, while Fpn1 expression was unaffected. Heph protein levels were significantly reduced (<90%) in cytosolic and membrane fractions of the KD cells. Ferrous iron oxidation measurements, using a transferrin‐coupled assay, showed that Heph KD had very little effect on membrane and cytosolic ferroxidase (FOX) activity. Additional studies showed for the first time that Heph was detected in early and late endosomes isolated from Caco‐2 cells, fractions in which Rab7 was also detected (an endosomal marker). This investigation has thus provided further evidence that Heph is not the sole FOX in mammalian intestinal epithelial cells.Grant Funding Source: Supported by NIH Grant, 2R01DK074867‐07A1