Silencing of the Menkes copper-transporting ATPase (Atp7a) gene increases cyclin D1 protein expression and impairs proliferation of rat intestinal epithelial (IEC-6) cells

Abstract

The Menkes copper-transporting ATPase (Atp7a) has dual roles in mammalian enterocytes: pumping copper into the trans-Golgi network (to support cuproenzyme synthesis) and across the basolateral membrane (to deliver dietary copper to the blood). Atp7a is strongly induced in the rodent duodenum during iron deprivation, suggesting that copper influences iron homeostasis. To investigate this possibility, Atp7a was silenced in rat intestinal epithelial (IEC-6) cells. Irrespective of its influence on iron homeostasis, an unexpected observation was made in the Atp7a knockdown (KD) cells: the cells grew slower (∼40% fewer cells at 96h) and were larger than negative-control shRNA-transfected cells. Lack of Atp7a activity thus perturbed cell cycle control. To elucidate a possible molecular mechanism, expression of two important cell cycle control proteins was assessed. Cyclin D1 (CD1) protein expression increased in Atp7a KD cells whereas proliferating-cell nuclear antigen (PCNA) expression was unaltered. Increased CD1 expression is consistent with impaired cell cycle progression. Expression of additional cell proliferation marker genes (p21 and Ki67) was also investigated; p21 expression increased, whereas Ki67 decreased, both consistent with diminished cell growth. Further experiments were designed to determine whether increased cellular copper content was the trigger for the altered growth phenotype of the Atp7a KD cells. Copper loading, however, did not influence the expression patterns of CD1, p21 or Ki67. Overall, these findings demonstrate that Atp7a is required for normal proliferation of IEC-6 cells. How Atp7a influences cell growth is unclear, but the underlying mechanism could relate to its roles in intracellular copper distribution or cuproenzyme synthesis.