Menkes Copper ATPase (Atp7a) Is a Novel Metal-responsive Gene in Rat Duodenum, and Immunoreactive Protein Is Present on Brush-border and Basolateral Membrane Domains

Abstract

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964 -G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic Atp7b gene expression does not change with iron deficiency, suggesting that liver copper excretion is not altered. We have developed polyclonal antibodies against rat ATP7A, and we demonstrate the specificity of the immunogenic reaction. We show that the ATP7A protein is present on apical domains of duodenal enterocytes in control rats and on brush-border and basolateral membrane domains in iron-deprived rats. This localization is surprising, as previous in vitro studies have suggested that ATP7A traffics between the trans-Golgi network and the basolateral membrane. We further demonstrate that ATP7A protein levels are dramatically increased in brush-border and basolateral membrane vesicles isolated from iron-deficient rats. Other experiments show that iron refeeding partially corrects the hematological abnormalities seen in iron-deficient rats but that it does not ameliorate ATP7A protein induction, suggesting that Atp7a does not respond to intracellular iron levels. We conclude that ATP7A is involved in copper loading observed during iron deficiency and that increased intestinal copper transport is of physiological relevance, as copper plays important roles in overall body iron homeostasis.Iron is a component of many proteins that are involved in cellular growth and metabolism. Complex homeostatic mechanisms exist to ensure that adequate iron is available for normal physiological functions while simultaneously avoiding excessive iron accumulation in body tissues. Free iron is highly reactive and is prone to catalyze the formation of toxic oxygen radicals via the Fenton reaction (1). Iron overload resulting from genetic diseases leads to hepatic fibrosis and cancer, diabetes mellitus, neurodegenerative disorders, and cardiomyopathy (2). Furthermore, iron deficiency is a major clinical issue, likely afflicting billions of people worldwide, including a large number of people in developed countries such as the United States (3-5). Iron deficiency anemia due to low dietary Fe intake results in impaired neurological development and decreased metabolic rate. To avoid iron deficiency and iron overload, iron balance is maintained via precise regulation of intestinal iron transport, which is the site for hormonal regulation of overall body iron homeostasis (6, 7). This is especially critical for this important trace mineral, as iron excretion is an unregulated, inefficient process.Within the past few years, several proteins involved ...

Serum ceruloplasmin protein expression and activity increases in iron-deficient rats and is further enhanced by higher dietary copper intake

“…50 Interestingly, under low iron conditions in rats, Atp7a expression is strongly induced 3,4 and the protein is present on the basolateral membrane of enterocytes, suggesting enhanced copper uptake. 9 After export of copper from enterocytes, binding to albumin or other serum proteins occurs for delivery and transport into hepatocytes by Ctr1. It is tempting to speculate that the induction of Atp7a (and presumed increased copper export from enterocytes) increases copper delivery to hepatocytes, secondarily leading to increased holo-Cp production.…”

“…Several key genes encoding iron transport-related proteins are strongly induced during iron deprivation, [3][4][5] some by posttranscriptional stabilization of mRNA transcripts, 6 and others via transcriptional induction mediated at least in part by a hypoxia-responsive trans-acting factor, hypoxia-inducible factor (HIF) 2␣. 7,8 In addition, genes related to intestinal copper homeostasis are induced during iron deprivation in rats, 4,9 suggesting that copper is important in the physiologic response of the intestinal epithelium to iron deficiency. Copper levels are known to increase in the liver and serum of iron-deficient mammals, [10][11][12] further hinting at an important physiologic role for copper during iron deprivation.…”

Discovery of a cytosolic/soluble ferroxidase in rodent enterocytes

“…Thirty micrograms of cytosolic or membrane proteins were resolved by SDS-7.5% PAGE, blotted, and immunoprobed as described earlier (21). The anti-Atp7a antibody (called "54-10") (20,22) and anti-ZnT1 (a kind gift from Robert Cousins, University of Florida, Gainesville, FL) (23) are well validated. Rabbit polyclonal anti-Heph Ab was from GeneTex (cat.…”

Dynamics of endogenous ATP7A (Menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites

“…Recently, endogenous ATP7A was reported to be in the duodenal brush border and basolateral membranes of rats maintained for 6 wk on an iron-deficient diet (49). Both indirect immunofluorescence and subcellular fractionation were used.…”