Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models.
Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting<lactating<suckling), indicating a link between function and copper requirement. Based on previous data showing a function for CTR1 in copper uptake, we have concluded that under the influence of hormones and increased extracellular copper levels, CTR1 participates in uptake of copper by mammary epithelial cells, as a prerequisite for secretion of copper into milk.
Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Victoria 3121, Australia.
David
D
Freestone
Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Victoria 3121, Australia; Department of Pathology, the University of Melbourne, Parkville, Victoria 3010, Australia.
Michael A
MA
Cater
Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Victoria 3121, Australia.
M Leigh
ML
Ackland
Australian Synchrotron, Melbourne, Victoria 3068, Australia.
David
D
Paterson
Australian Synchrotron, Melbourne, Victoria 3068, Australia.
Daryl L
DL
Howard
Australian Synchrotron, Melbourne, Victoria 3068, Australia.
Martin D
MD
de Jonge
Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Victoria 3121, Australia. Electronic address: agnesm@deakin.edu.au.
Agnes
A
Michalczyk
D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003300:Copper / Q000378:metabolism* / Q000493:pharmacokinetics; D000079923:Copper Transporter 1; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D007774:Lactation / Q000378:metabolism*; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism
D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; C490079:SLC31A1 protein, human; D003300:Copper; D011388:Prolactin
CTR1; Copper; Human mammary gland; Lactation; Metallothionein; X-ray fluorescence microscopy