title: Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models.
abstract: Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.
authors.affiliation: Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125, Australia. agnes.michalczyk@deakin.edu.au.
authors.forename: David
authors.initials: D
authors.lastname: Freestone
authors.forename: Delphine
authors.initials: D
authors.lastname: Denoyer
authors.forename: Matthew
authors.initials: M
authors.lastname: Jakab
authors.forename: M
authors.initials: M
authors.lastname: Leigh Ackland
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cater
authors.forename: Agnes
authors.initials: A
authors.lastname: Michalczyk
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002570:Ceruloplasmin / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006728:Hormones / Q000494:pharmacology*; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice
chemical_list: D006728:Hormones; D003300:Copper; D002570:Ceruloplasmin