Differential effects of basolateral and apical iron supply on iron transport in Caco-2 cells

Abstract

Iron homeostasis in the human body is maintained primarily through regulation of iron absorption in the duodenum. The liver peptide hepcidin plays a central role in this regulation. Additionally, expression and functional control of certain components of the cellular iron transport machinery can be influenced directly by the iron status of enterocytes. The significance of this modulation, relative to the effects of hepcidin, and the comparative effects of iron obtained directly from the diet and/or via the bloodstream are not clear. The studies described here were performed using Caco-2 cell monolayers as a model of intestinal epithelium, to compare the effects of iron supplied in physiologically relevant forms to either the apical or basolateral surfaces of the cells. Both sources of iron provoked increased cellular ferritin content, indicating iron uptake from both sides of the cells. Supply of basolateral transferrin-bound iron did not affect subsequent iron transport across the apical surface, but reduced iron transport across the basolateral membrane. In contrast, the apical iron supply led to subsequent reduction in iron transport across the apical cell membrane without altering iron export across the basolateral membrane. The apical and basolateral iron supplies also elicited distinct effects on the expression and subcellular distribution of iron transporters. These data suggest that, in addition to the effects of cellular iron status on the expression of iron transporter genes, different modes and direction of iron supply to enterocytes can elicit distinct functional effects on iron transport.

Fig. 1

Expression of iron transport genes in Caco-2 cells over time following cell seeding in Transwell^® permeable support plates. Values shown indicate relative mRNA levels for a TfR-1, b DMT-1 and c FPN in Caco-2 cells determined at different days ranging from 10 to 21 post-seeding cultured with normal medium. Bars and error bars indicate mean + SD (n = 4 comprised of samples generated from 1 well from each of two separate plates set up in parallel in two independent experiments). Data were compared by one-way ANOVA with Tukey’s post hoc test. Asterisks indicate statistically significant differences between days (*p < 0.05, **p < 0.01 and ***p < 0.001)

Fig. 2

Effects of different forms of iron, supplied to Caco-2 cell monolayers via either the apical or basolateral surface, on intracellular ferritin levels. Values indicate ferritin concentrations determined in extracts derived from Caco-2 cell monolayers that had been established over 2 weeks following seeding in bicameral chambers before being swapped for one further week (days 14–21 following cell seeding) into medium prepared with metal-depleted FBS only (Control), medium prepared with metal-depleted FBS plus 30 μM holo-Tf added to the medium only on the basolateral side of the cells (30 μM holo-Tf) or medium prepared with metal-depleted FBS plus ferric NTA (FeNTA) at 2 or 10 μM added to the medium only on the apical side of the cells. The bars and error bars indicate mean values + SD (n = 8 comprised of 4 replicate wells for each treatment from each of two separate plates set up in parallel). Data were compared by one-way ANOVA with Tukey’s post hoc test. Asterisks indicate statistically significant differences between treatments (*p < 0.05 and ****p < 0.0001)

Fig. 3

Effects of treatment of Caco-2 cell monolayers with a basolateral supply of holo-Tf or an apical supply of ferric NTA on subsequent iron transport across the cell layer. Values shown indicate a total cellular iron uptake (sum of iron retained within the cells and iron transported from the apical to the basolateral medium) and b apical to basolateral iron transport of iron supplied to the apical side of the Caco-2 cell monolayer on day 21, following incubation from day 14 to 21 with medium containing metal-depleted FBS in the absence of any added iron (Control), presence of 10 μM ferric NTA added to the apical side of the cells only (10 μM FeNTA) or of 30 μM holo-Tf added to the basolateral side of the cells only (30 μM holo-Tf). Bars and error bars indicate mean + SD (n = 8 comprised of 4 replicate wells for each treatment from each of two separate plates set up in parallel). Data were compared by one-way ANOVA with Tukey’s post hoc test. Asterisks indicate statistically significant differences between treatments (*p < 0.05 and **p < 0.01)

Fig. 4

Effects of treatment of Caco-2 cell monolayers with a basolateral supply of holo-Tf or an apical supply of ferric NTA on mRNA levels for iron transport genes. Values shown indicate relative mRNA levels for a TfR-1, b DMT-1 and c FPN in Caco-2 cells determined at day 21 post-seeding following incubation from day 14 to 21 of confluent Caco-2 cell monolayers established in bicameral chamber with medium containing metal-depleted FBS in the absence of any added iron (Control), presence of 10 μM ferric NTA added to the apical side of the cells only (10 μM FeNTA) or of 30 μM holo-Tf added to the basolateral side of the cells only (30 μM holo-Tf). Bars and error bars indicate mean + SD (n = 10 comprised of samples generated from 1 or 2 wells for each treatment from each of three separate plates set up in parallel with a total of five samples from each of two independent experiments). Data were compared either by Kruskal–Wallis test with Dunn’s multiple comparison post hoc test (for DMT-1 and TfR-1 where at least one data set did not pass the D’Agostino and Pearson’s omnibus normality test) or by one-way ANOVA with Tukey’s post hoc test (for FPN). Asterisks indicate statistically significant differences between treatments (*p < 0.05, **p < 0.01 and ***p < 0.001)

Fig. 5

Effects of treatment of Caco-2 cell monolayers with a basolateral supply of holo-Tf or an apical supply of ferric NTA on whole-cell and cell surface levels of proteins responsible for transport of iron across the basolateral membrane of enterocytes. Relative protein levels for whole-cell TfR-1 (a), cell surface TfR-1 (b), whole-cell FPN (c) and cell surface FPN (d) in Caco-2 cells determined at day 21 post-seeding following incubation from day 14 to 21 of confluent Caco-2 cell monolayers established in bicameral chamber with medium containing metal-depleted FBS in the absence of any added iron (Control), the presence of 10 μM ferric NTA added to the apical side of the cells only (10 μM FeNTA) or 30 μM holo-Tf added to the basolateral side of the cells only (30 μM holo-Tf). Insets above each graph depict and an example of the one of the western blots from which the data for the graph were generated. Bars and error bars in the graphs indicate mean + SD (n = 6 comprised of samples generated from 2 wells for each treatment from each of 3 separate plates set up in parallel). Data were compared by one-way ANOVA with Tukey’s post hoc test. Asterisks indicate statistically significant differences between treatments (*p < 0.05 and **p < 0.01)