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ATP7a Silencing Impairs Iron Transport in Fully‐Differentiated Rat Intestinal Epithelial (IEC‐6) Cells
Author(s) -
Ha JungHeun,
Gulec Sukru,
Doguer Caglar,
Flores Shireen,
Han Yi,
Wang Xiaoyu,
Collins James
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.263.5
Subject(s) - dmt1 , atp7a , ferroportin , metallothionein , homeostasis , hepcidin , iron deficiency , medicine , chemistry , endocrinology , hemoglobin , weanling , transferrin receptor , downregulation and upregulation , anemia , biology , atpase , transferrin , biochemistry , transporter , enzyme , gene
Intestinal iron absorption is tightly regulated since no active excretory pathways for iron exist in mammals. Iron homeostasis isintertwined withthat of copper (Cu), but specific molecular interactions are yet to be defined. To further evaluate iron‐copper interplay in the mammalian intestine, low, control, or high‐iron diets (3, 50and 10,000 ppm, respectively) were fed to weanling rats ad libitum for 5 weeks. Rats fed the low‐iron diet developed iron‐deficiency anemia, and genes involved in duodenal Fe homeostasis increased significantly as compared to the other groups. Interestingly, genes encoding proteins involved in Cu homeostasis were also upregulated in the low Fe group, including copper‐transporting ATPase 1 (ATP7a) and metallothioneinLow iron Control iron High iron Hemoglobin 2.8 (0.29)**** 13.09 (0.64) 13.14 (0.64) Hematocrit (%) 14.05 (2.58)**** 50.61 (4.78) 50.47 (1.15) Atp7a mRNA 6.34 (2.69)**** 1.03 (0.28) 2.18 (1.31) Metallothionein 1a 12.59 (15.81)* 1.85 (2.40) 1.30 (0.94) Dmt1 mRNA 179.89 (84.5)**** 1.07 (0.42) 1.67 (0.88) Dcytb mRNA 6939.76 (4386.76)**** 1.70 (1.88) 17.56 (21.17) Ferroportin1 mRNA 3.95 (1.55)**** 1.15 (0.62) 1.66 (1.13) Transferrin receptor 1 mRNA 10.67 (2.83)**** 1.09 (0.47) 0.71 (0.25)We thus hypothesized that ATP7a function is important for intestinal iron metabolism. To test this postulate, we blocked ATP7a expression in rat IEC‐6 cells by shRNA‐mediated gene silencing. Notably, ATP7a knock down (KD) impaired Fe uptake and efflux in fully‐differentiated cells. Moreover, ATP7a KD decreased doudenal cytochrome b and ferroportin 1 mRNA expression, while conversely, hephaestin (Heph) gene expression increased significantlyConsistent with Heph induction, ferroxidase (FOX) activity in total cell lysates increased in the KD cellsThese observations demonstrate that ATP7a activity is necessary for iron absorption in IEC‐6 cells. Altered expression of iron transport‐related genes in the KD cells could be the underlying molecular mechanism by which ATP7A alters iron flux. Furthermore, increased Heph expression and FOX activity may be a compensatory response to increase iron transport.