Evidence there are at least three ways for copper to be taken up by enterocytes across the brush border, as investigated in the Caco2 cell model

The specifics of intestinal copper absorption by enterocytes and the effects of copper and iron status on this process are still not completely understood. The Caco2 polarized cell culture models for enterocytes was used to determine whether copper transporter 1 (CTR1) participates in Cu uptake across the brush border membrane and is located on that side of the cell monolayer; whether divalent metal transporter 1 (DMT1), a chloride dependent transporter, and another as yet unknown transporter are also involved; and whether the status of Cu and Fe of the cells has any effect. Quantitative PCR analysis of Caco2 cells grown on six‐well tissue culture plates showed that CTR1 and DMT1 mRNA were expressed in about equal proportions relative to 18S rRNA. Both DMT1 and CTR1 expression increased overall when the cell culture medium was supplemented with 1 uM Cu(II) histidine for 24 hours. Potential effects of changes in iron status are under investigation. To test if increased mRNA expression resulted in a greater rate of copper uptake, cells were seeded on bicameral chambers and allowed to polarize. Transepithelial electrical resistance was measured to determine the formation of tight junctions. Cells were then untreated or pretreated with 5uM copper to induce copper excess or triethylenetetramine (TETA) to induce copper deficiency. Rates of uptake of Cu(I) (5uM) were measured by following uptake of 67 Cu into cells and the basolateral fluid over 1h, after application to the apical (brush border) side of the monolayer, in serum‐free HEPES buffered medium. Copper supplementation did not enhance the rate of copper uptake, but copper deficiency significantly reduced it by 20–35%. Supplementation with 5uM Fe(II) (as the nitrilotriacetate complex) to induce iron excess, or 10 uM desferrioxamine (to induce deficiency) had little to no effect on the ability of the monolayers to take up copper across the brush border. To check for activity of a chloride dependent transporter, uptake of 67 Cu(I) across the apical portion of the monolayer was measured in HEPES buffered medium in which Na 2 SO4, MgSO 4 , and K 2 SO 4 was substituted for NaCl, MgCl 2 , and KCl. Cells were also incubated with and without silver in order to also determine the importance of CTR1 to dietary copper uptake. Generally, copper uptake was reduced by a small amount by 10–20% when chloride was replaced with sulfate. Addition of Ag(I) (50 uM) reduced uptake 20–50%, the largest effect being in the presence of silver and sulfate, thus leaving at least half of the uptake occurring through other means. The assumed role of a reductase in copper uptake was also examined. By qPCR, we found that Caco2 cells expressed STEAPs 2 and 3 but not 4 mRNA, and did not express duodenal cytochrome B. Measurements of Cu(II) reductase activity by the method of Wyman et al, using 50 uM Cu(II)‐NTA and 200 uM bathocuproine disulfonate, indicated the presence of cell surface reductase activity. Efforts to knock out DMT1 and STEAP2 by CRISPR/Cas9 are currently underway to see their impact on copper absorption, as is confocal microscopy to definitively locate CTR1. We conclude that copper absorption by enterocytes is more complex than generally assumed, and there may be an as yet unidentified copper uptake transporter in the brush border membrane of enterocytes.