Expression of Menkes disease gene in mammary carcinoma cells

Abstract

Two P-type ATPases, MNK and WND were recently shown to be defective in the human disorders of copper transport, Menkes disease and Wilson disease respectively. These proteins are important in copper homeostasis but their full physiological function has not been established. This study uses the human breast carcinoma line, PMC42, to investigate copper transport in the mammary gland. Northern blot analysis indicated that both MNK and WND mRNA are expressed in these cells. Western blot analysis with an MNK-specific antibody demonstrated a band of approx. 178 kDa, close to the expected size of 163 kDa. Treatment of PMC42 cells with lactational hormones (oestrogen and progesterone for 3 days followed by dexamethasone, insulin and prolactin for a further 3 days) did not produce an obvious increase in MNK expression as measured by Northern and Western blots. By using indirect immunofluorescence with the MNK antibody, the intracellular distribution of MNK was found to be predominantly perinuclear, consistent with Golgi localization. Punctate staining was also seen in a smaller proportion of cells, suggesting that some MNK is associated with endosomes. Treatment of PMC42 cells with lactational hormones increased the intensity of the perinuclear and punctate fluorescence. Exposure of cells to 100 mM copper resulted in the dispersion of the fluorescence towards the periphery of the cell. The results suggest a role for MNK in the secretion of copper into milk and that PMC42 cells are a valuable model for investigating the detailed cellular function of MNK and WND.

Cloning and Characterization of an Atypical Type IV P-type ATPase That Binds to the RING Motif of RUSH Transcription Factors

“…Menkes-and Wilson-ATPases have been implicated in human disorders of copper homeostasis. In the case of the Menkes P-type ATPase, treatment of human breast carcinoma cells, PMC42, with a combination of estrogen, progesterone, and prolactin increased perinuclear (Golgi) and punctate (endosome) protein, as measured by indirect immunofluorescence (57). However, Northern analysis failed to show an effect of hormones on mRNA expression.…”

Multiple Menkes copper ATPase (Atp7a) transcript and protein variants are induced by iron deficiency in rat duodenal enterocytes

“…Previously, Reddy et al [10] used a 530 bp oligonucleotide homologous to exon 23 as a probe and identified only one hybridization band at ~5.5 kb in human cells. Moreover, Ackland et al [11], generated probes from the “N-terminal” region of the Atp7a transcript and reported a single hybridization band at ~8.5 kb in human breast cancer cells. In the current study, full-length Atp7a probes also hybridized with transcripts in RNA isolated from rat IEC-6 cells (Fig.…”

The multi-layered regulation of copper translocating P-type ATPases

“…The Tx mutation prevents WND Cu-translocating activity and WND exocytosis from the TGN, causing Cu accumulation within the tissue and reduced Cu secretion into milk (Michalczyk et al 2000;Rouch 1983;Voskoboinik et al 2001a). It is now clear that WND plays a physiological secretory role by delivering Cu to infants in milk and ceruloplasmin (Ackland et al 1999;Ackland et al 1997;Kelleher and Lonnerdal 2006). MNK is responsible for direct Cu export in response to lactation, where hormonal induction by estrogen, progesterone, insulin and prolactin stimulate the expression and basolateral localization of MNK and the Cu uptake protein Ctr1 to perform a homeostatic function in the mother's tissue (Ackland et al 1997;Llanos et al 2008).…”

“…It is now clear that WND plays a physiological secretory role by delivering Cu to infants in milk and ceruloplasmin (Ackland et al 1999;Ackland et al 1997;Kelleher and Lonnerdal 2006). MNK is responsible for direct Cu export in response to lactation, where hormonal induction by estrogen, progesterone, insulin and prolactin stimulate the expression and basolateral localization of MNK and the Cu uptake protein Ctr1 to perform a homeostatic function in the mother's tissue (Ackland et al 1997;Llanos et al 2008). Studies in cultured placental cells support this finding, where insulin and estrogen treatment of cultured placental JEG-3 cells promotes MNK trafficking toward the basolateral surface for enhanced Cu export, in the absence of added Cu.…”