Research article
Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models

https://doi.org/10.1016/j.jnutbio.2013.11.011Get rights and content

Abstract

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.

Introduction

Copper in breast milk is critical for the postnatal growth and development of mammalian infants. The importance of copper at this stage is clearly illustrated by the toxic milk (tx) mouse model, where a defective mAtp7b (P1B-type ATPase) protein impedes supply of copper into the dam’s milk causing the suckling pups to become copper deficient; leading to their death after approximately 2 weeks of age [1], [2]. During lactation copper levels fluctuate greatly in the mother’s blood independent of dietary copper uptake [3]. Concurrently, the rate of copper absorbed by the mammary gland can increase up to 20-fold during lactation [4]. Mammalian milk copper concentrations are highest the first week after birth (humans; 0.4–0.6 mg/L, rodents; 3.2–6.4 mg/L) then decrease substantially (~50%) as lactation progresses (humans; 0.2–0.3 mg/L, rodents; 0.9–2.1 mg/L) [5], [6], [7], with copper secretion into milk from the mammary gland stimulated by suckling [8]. Copper secretion into milk is regulated to not expose the suckling infant to excess or deficient levels, and thus allowing for optimal growth and development.

Until pregnancy, mammary epithelial cells remain dormant. Hormonal changes in pregnancy play an important role in modification of the mammary gland, initiating the growth and proliferation of both ducts and alveoli [9]. Estrogen and progesterone regulate the differentiation and development of mammary epithelial cells into secretory alveoli. Postpartum, estrogen and progesterone levels then reduce to pre-pregnancy levels, initiating the secretion of milk [10], [11]. Other hormones including dexamethasone, insulin and prolactin are crucial for the differentiation of mammary glands and in the induction of lactation [10], [12]. Interestingly, circulating prolactin concentrations in maternal blood positively correlate with mammary gland copper transport and prolactin has been shown to modulate the three main copper-transport proteins, mCtr1 (copper transporter 1), mAtp7a (P1B-type ATPase) and mAtp7b in mouse mammary epithelial cells [8]. However, the molecular mechanisms that regulate mammary gland copper uptake and secretion into milk, including interplay between the copper-transport proteins, are not well understood. In the mammary gland, the high affinity copper uptake protein, CTR1, localises along the basolateral membrane, positioned to mediate copper uptake from maternal blood directly into mammary epithelial cells [8], [13], [14]. CTR1 is a 190-amino-acid glycosylated protein that is approximately 35 kDa and has three transmembrane domains with an exofacial N-terminus and a cytosolic C-terminus [15]. Electron crystallography revealed that three CTR1 proteins can bind together to form a radially-symmetrical conical-shaped channel that is widest on the cytosolic side [16]. The exofacial N-termini coordinate Cu(II) from the extracellular environment. Subsequently, Cu(II) is reduced to Cu(I) and passively transported through the channel to the C-termini, where it becomes available to copper chaperones like ATOX1 [17], [18]. In some mammalian cells (e.g., HEK293) elevated extracellular copper (1–100 μM) stimulates rapid internalisation of CTR1 from the plasma membrane [19], while in other cells (e.g., HeLa and Caco-2) CTR1 localisation remains unreceptive to copper [20]. Internalisation of CTR1 could provide a quick and reversible mechanism for the regulation of cellular copper uptake.

In this study we investigated the regulation of CTR1 in cellular models of the resting, lactating and suckling mammary gland. A disadvantage of many mammary epithelial cell lines utilised for in vitro studies is the lack of capacity to differentiate. Previous studies demonstrated that mammary epithelial PMC42-LA cells grown on plastic retain breast-specific features including lipid granules, swollen endoplasmic reticulum and large secretory vacuoles [21], [22]. Additionally, when grown on specialised extracellular matrixes (e.g., derived from Engelbreth Holm-Swarm mouse sarcoma) PMC42-LA cells can form organoid structures similar to alveoli of the mammary gland, where secretory cells are arranged around a central lumen [23]. These cells develop a phenotype more representative of differentiated luminal epithelial cells, including the presence of tight junctions and microvilli, basally located nuclei and the presence of lipid granules [23]. Furthermore, differentiated PMC42-LA cells also express proteins found in milk and upon stimulation with lactational hormones express β-casein; a marker often used to identify fully differentiated lactating mammary epithelial cells [23], [24], [25]. Using PMC42-LA–based models representing resting, lactating and suckling mammary glands, we have demonstrated that both the degree of mammary epithelial differentiation and the level of extracellular copper greatly impact upon CTR1 expression and plasma membrane association. Furthermore, copper resides predominantly in organoid structures with the level rising as PMC42-LA cells become progressively more specialised towards suckled mammary gland. How CTR1 may aid the movement of copper from maternal blood into milk to sustain the rapid growth of the neonate is discussed.

Section snippets

Cell culture

Breast adenocarcinoma PMC42-LA cells, a variant of the PMC42 line, were originally derived from a pleural effusion [21] and were cultured at 37ºC (5% CO2) in RPMI 1640 medium (Thermo Fisher Scientific; Melbourne, Australia) supplemented with 10% FBS (Bovogen; Melbourne, Australia). PMC42-LA cells were differentiated on porous Transwell filters (BD Falcon; Sydney, Australia) coated thinly with undiluted extracellular matrix gel from Engelbreth Holm-Swarm mouse sarcoma (EHS matrix)

Copper modulates CTR1 expression and plasma membrane association in undifferentiated PMC42-LA cells

CTR1 expression and subcellular localisation was initially investigated in undifferentiated PMC42-LA cells grown in media supplemented with various concentrations (0–50 μM) of copper (CuCl2) for 3 days (Fig. 1). A concentration of 5 μM copper was use to match the normal level of exchangeable copper in women’s plasma (between 2.7–7.7 μM) [33], [34], while 50 μM copper was added to analyse the effect of high copper. Additionally, we cultured undifferentiated PMC42-LA cells with medium

Discussion

As CTR1 is postulated to mediate copper absorption from the maternal bloodstream into mammary epithelium for incorporation into milk [8], we therefore investigated CTR1 protein regulation in response to hormonal treatments simulating the different lactational states. We utilised previously developed culture models of resting and lactating mammary gland [23], [24], [25], in combination with a novel model representing the suckled state, to gain insights into CTR1 regulation and mammary epithelial

Acknowledgments

Part of this research was undertaken on the XFM beamline at the Australian Synchrotron, Victoria, Australia. Dr Cater is funded by a Movember Young Investigator Grant awarded through Prostate Cancer Foundation of Australia’s Research Program and by the National Health and Medical Research Council of Australia (NHMRC).

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