Biochemical and Biophysical Research Communications
Copper transport during lactation in transgenic mice expressing the human ATP7A protein
Section snippets
Materials and methods
Animals. All animal experiments were performed in accordance with the Deakin University Animal Welfare Committee regulations, approval numbers A20/2001 and A41/2004. Non-transgenic and transgenic mice, in the C57BL/6 background, were bred and maintained on a 12-h light/12-h dark cycle, and given deionised water and standard non-purified diet (Barastoc, Ridley AgriProducts, Australia) ad libitum during pregnancy and lactation. Mammary gland, liver, brain, and kidney from dams were removed at
Establishment of a homozygous ATP7A transgenic mice colony
A homozygous ATP7A transgenic mice colony from line T22#2 [19] was established by mating heterozygote mice which were identified by duplex PCR of tail DNA as described in Materials and methods. The homozygous T22#2 ATP7A transgenic mice, from here on referred to as ‘transgenic mice’, were subsequently maintained by brother sister mating. The homozygous male and female mice displayed no overt phenotype.
Expression of ATP7A in mammary gland tissue
The expression levels of ATP7A and the endogenous Atp7a mRNA and ATP7A protein in mammary
Discussion
In the mammary epithelial cells ATP7A was primarily associated with the basolateral membrane of the epithelial cells, but some cytoplasmic staining was evident. No ATP7A was observed at the apical surface of the cell in contrasts to the report of apical localization of the protein in lactating rat mammary gland [17]. The discrepancy could be due to the species difference, suckling stimulation, or stage of lactation. The basolateral localization suggests that ATP7A is not primarily involved in
Acknowledgments
This work was supported by the National Institutes of Health under Grant R01 HD46949-01. We thank Bi-Xia Ke for assistance with atomic absorption spectroscopy.
References (28)
The molecular basis of copper transport diseases
Trends Mol. Med.
(2001)- et al.
Copper exposure induces trafficking of the menkes protein in intestinal epithelium of ATP7A transgenic mice
J. Nutr.
(2005) - et al.
ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion
Gastroenterology
(2006) - et al.
Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion
Gastroenterology
(2000) - et al.
Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells
Placenta
(2006) - et al.
Correction of a mouse model of Menkes disease by the human Menkes gene
Biochim. Biophys. Acta
(2006) - et al.
Milk ceruloplasmin and its expression by mammary gland and liver in pigs
Arch. Biochem. Biophys.
(2000) - et al.
Copper incorporation into ceruloplasmin in rat livers
Biochim. Biophys. Acta
(1995) - et al.
Functional studies on the Wilson copper P-type ATPase and toxic milk mouse mutant
Biochem. Biophys. Res. Commun.
(2001) - et al.
The copper transporter CTR1 provides an essential function in mammalian embryonic development
Proc. Natl. Acad. Sci. USA
(2001)
Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development
Proc. Natl. Acad. Sci. USA
Toxic milk, a new mutation affecting copper metabolism in the mouse
J. Hered.
Defective trafficking of the Wilson disease protein ATP7B in the mammary gland of the toxic milk mouse and the effects of copper supplementation
Biochem. J.
The toxic milk mouse is a murine model of Wilson disease
Hum. Mol. Genet.
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Ceruloplasmin gene expression profile changes in the rat mammary gland during pregnancy, lactation and involution
2017, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :During lactation, ATP7B-mRNA levels changed with sCp-mRNA levels. Considering that: (1) the ATP7A/B proteins were discovered using methods of immunohistochemistry, immunofluorescence, and WB analysis of the total cellular extracts in pregnant and lactating MG [56,57]; (2) they were redistributed throughout the cytoplasm of the luminal epithelial cells under the action of high copper concentrations or hormone induced lactation; (3) ATP7A and ATP7B polypeptides can be localized in Golgi complex, endosomal compartment, secretor vesicles, plasma membrane, and associated with lysosomes [7,58] and (4) as components of these organelles they can redistribute from the perinuclear space to the plasma membrane and back again. We used high purity fractions of the plasma membrane to examine whether there was a relationship between the localization of ATPases on the membrane and the MG development cycle.
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2015, Knobil and Neill's Physiology of Reproduction: Two-Volume SetCopper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models
2014, Journal of Nutritional BiochemistryCitation Excerpt :Cells grown on Transwell inserts with EHS matrix were fixed in 4% (w/v) paraformaldehyde for 10 min, permeabilised with 3% (v/v) Triton X-100 for 10 min and blocked with 3% (w/v) BSA for 1 h. The CTR1 antibody was raised in rabbits against the first 98 amino acids of the human CTR1 protein [14,26,27]. Rabbit anti-CTR1 was diluted (1/1000) in 1% BSA before being applied to cells overnight at 4°C.
Human copper homeostasis: a network of interconnected pathways
2010, Current Opinion in Chemical BiologyMammalian copper-transporting P-type ATPases, ATP7A and ATP7B: Emerging roles
2010, International Journal of Biochemistry and Cell BiologyCitation Excerpt :In the mammary gland, ATP7A and ATP7B are expressed in luminal epithelial cells and their levels and/or distribution respond to lactational hormones that stimulate an increased flux of Cu through the mammary gland (La Fontaine and Mercer, 2007; Michalczyk et al., 2008). Here ATP7B transports Cu across the apical surface implicating it as the major means of Cu secretion into milk during lactation; whereas the basolateral location of Atp7a in transgenic mice suggests that ATP7A may serve a protective role to export excess Cu from the mammary gland back into the circulation (Llanos et al., 2008). ATP7A is widely expressed throughout the CNS and the elevation of Cu in vascular endothelial cells of the brains of MD patients indicates a role for this ATPase in Cu transport across the blood–brain barrier.