Silencing of the Menkes Copper‐Transporting ATPase (Atp7a) in Intestinal Epithelial Cells Impairs Cell Growth and Cell Cycle Control

The Menkes copper‐transporting ATPase (Atp7a) is induced in the rodent duodenum during iron deficiency, suggesting that copper influences iron homeostasis. To understand the role of Atp7a in intestinal iron & copper metabolism, we generated an Atp7a knockdown (KD) IEC‐6 cell line using shRNA technology. Atp7a protein was reduced >;70% and KD cells accumulated copper, consistent with defective copper efflux mediated by Atp7a. Unexpectedly, Atp7a KD impaired growth rate as compared to negative control shRNA‐transfected cells (~40% at 96 hours; p<0.0001). Further experiments were thus designed to elucidate the role of Atp7a in cell growth. Western blot analysis demonstrated that cyclin D1 (CD1) expression increased in KD cells while PCNA expression was unchanged. CD1 induces cell cycle arrest while PCNA promotes cell division. This suggested that Atp7a KD perturbed progression through the cell cycle. CD1 expression was not further enhanced by copper loading, implicating Atp7a in regulation of cell growth independent of its documented role in maintaining cellular copper homeostasis. Subsequent experiments showed that expression of additional markers of cell cycle control (p53, p21 & Ki67) was consistent with decreased cell growth. Overall, these novel findings support the hypothesis that Atp7a function is important for regulation of intestinal epithelial cell proliferation and differentiation. Grant Funding Source : NIH