Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Abstract

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Fig. 1

Transgene expression in the mammary gland. Representative Western blot of 50 μg mammary gland protein extracts from three non-transgenic (Non-tg) and three transgenic (Tg) females. The R17-BX antibody detected both ATP7A and Atp7a (178 kDa). Keratin 18, a protein expressed in single layer epithelial tissues was used as a loading control.

Fig. 2

Endogenous expression of other copper-related genes. (A) Relative levels of endogenous Atp7a, Atp7b, Cp, and Ctr1 mRNA in mammary gland of transgenic (Tg) mice, n = 5 compared to non-transgenic (Non-tg) mice, n = 9 normalized against β-actin. Real time quantitative PCR data represent mean fold change ± SD from three duplicate determinations. *Significant transgene effect, P = 0.012. (B) Representative Western blots of 50 μg mammary gland protein extracts from three Non-tg and three Tg females. Keratin 18, a protein expressed in single layer epithelial tissues was used as a loading control.

Fig. 3

Immunofluorescence analysis of mammary gland tissue. Tissue sections were processed as described in Materials and methods and proteins were detected by indirect immunofluorescence with R17-BX (green) and β-integrin (red) (top panels), R17-BX (green) and anti-Atp7b (red) (middle panels), and R17-BX (green) and anti-hCTR1 antibodies (red) (bottom panels) using a Leica confocal microscope.

Fig. 4

Tissue copper concentration in lactating non-transgenic and transgenic mice. Copper concentrations in tissues from non-transgenic (Non-tg), n = 9 and transgenic (Tg) mice, n = 5 were determined by atomic absorption spectroscopy. Data are means ± SD. *Significant transgene effect, P = 0.023 for mammary gland (MG), P < 0.001 for brain, P = 0.039 for kidney, and P < 0.001 for stomach contents of pups.