Enterocyte cells are unique cell types in the intestine due to the interaction between diet and the internal circulatory system of the body. Thus, the dietary and blood-derived stimulus might play a role in enterocyte nutrient metabolism. Copper and iron have physiological interactions in enterocyte cells during IDA. In many mammalian species, iron deficiency leads to elevated Cu levels in enterocyte16, serum17, and liver18, indicating that copper might influence intestinal iron metabolism. Iron and copper interactions have been investigated in brindled mice (MoBr/y) and researchers observed no perturbation in iron absorption during anemia9. In this study, the enterocyte copper level of mutant mice was high due to nonfunctional ATP7A protein. In contrast, these mice were systemic copper-deficient; thus, enterocyte cells were exposed to two different copper levels. Furthermore, it was observed that dietary-induced iron deficiency upregulated mRNA expressions of iron-regulated genes, including DMT1, TFR, and ATP7A, but high enterocyte copper level did not affect the gene regulations suggesting that blood Cu level might be an important dietary factor for gene regulations in enterocyte cells during IDA. Thus, we grew Caco-2 cells on a bicameral cell culture plate to mimic the human intestine system to test whether dietary (apical side) or blood copper (basolateral side) are involved in iron-dependent genes regulation during IDA.
In the current study, we selected marker genes that were regulated by iron deficiency anemia including iron transporters (DMT1, TFR and FPN) and iron-dependent hypoxic genes (ANKRD37 and EGLN3). Furthermore, MT1A mRNA level was used to control intracellular mineral uptake since MT1A is regulated by intracellular Cu and Zn, respectively19,20. Our results showed that Cu and Cu with Fe in the basolateral side of the cells reduced marker genes mRNA levels (DMT1, FPN, TFR1, and ANKR37) compared to the apical Cu and Cu with Fe treatment group during IDA. This suggests that Cu sensing of the basolateral side of the cells might be different from Cu sensing of the apical side. Cu regulates the MT1A mRNA levels and its level is correlated by elevated intracellular Cu20. We observed that MT1A mRNA levels were significantly induced when Cu and Cu with Fe were given to the basolateral side of the cells suggesting that the level of Cu in the blood might be a compelling factor for the regulation of iron deficiency-related genes in the intestine. Furthermore, the basolateral side of the enterocyte cells may play a role in Cu sensing. The importance of the molecular and genetic interactions between basolateral and apical sides of polarized enterocyte cells has been indicated in different studies. It was shown that glucose treatment in the basolateral side of the Caco-2 cells induced apical cholesterol uptake and the mRNA levels of the cholesterol transporter gene21, indicating that basolateral signaling influences fatty acid metabolism through apical side of enterocyte cells. Moreover, Han et al. (2002)22 showed that both apical and basolateral Cu treatment increased Fe uptake in non-anemic Caco-2 cells. Moreover, Cu treatment of apical and basolateral sides also induced DMT1, TFR, and FPN mRNA levels compared to the control group. All together results suggest that basolateral and apical sides of enterocyte cells might have a different physiological response regarding the same nutritional stimulus.
Caco-2 cells can differentiate on the regular tissue culture plate when they are grown 21 post-confluent days. Differentiation of Caco-2 cells gives different genetic responses for a variety of cellular physiological pathways15. However, polarization of those cells on the insert system might be another factor that can influence genetic regulation. Thus, we also grew Caco-2 cells on regular tissue culture plates at the same time that we performed bicameral cell culture experiments. We observed similar results for marker genes regulation between apical side Cu treatment groups and Cu treatment on Caco-2 cells that were grown on tissue culture plates. However, basolateral Cu treatment affected regulations of DMT1, FPN, MT1A, ANKRD37 genes compared to results from the cells that were grown on tissue culture plates. Our results suggest that the polarization of the Caco-2 cells might be an important factor for gene regulation in terms of Cu treatment in our study. It might be better to account polarization of the Caco-2 cells for gene regulation or whole-genome array studies.