Assessment of ORAI1-mediated basal calcium influx in mammary epithelial cells
© Ross et al.; licensee BioMed Central Ltd. 2013
Received: 20 November 2013
Accepted: 9 December 2013
Published: 20 December 2013
The entry of calcium ions into mammary gland epithelial cells is one of the least well-understood processes in the transport of calcium into milk during lactation. The store-operated calcium entry channel ORAI1, has been suggested as a potential mechanism for the entry of Ca2+ into mammary gland epithelial cells from the maternal blood supply during lactation. The down regulation of the canonical ORAI1 activator STIM1 during lactation suggests that other known ORAI activators such as STIM2 and SPCA2 may be important during lactation.
Differentiation of HC11 mammary gland epithelial cells was associated with enhanced basal Ca2+ influx. Silencing of Orai1 abolished this enhancement of Ca2+ influx. Stim2 had a modest effect on Ca2+ influx in this in vitro model of lactation, whereas Stim1 and Spca2 silencing had no effect. Despite pronounced increases in Spca2 mRNA during lactation there was no change in the generation of the alternative splice product generated by Mist1, which increases during lactation.
These studies support the hypothesis that lactation is associated with a remodelling of Ca2+ influx and this is associated with enhancement of basal Ca2+ influx. This enhanced Ca2+ influx appears to occur through the calcium channel Orai1.
Lactation is the result of the finely orchestrated differentiation of mammary epithelial cells that gives them the ability to secrete milk. Mammary epithelial cells are unique in their ability to differentiate into lactogenic phenotypes and then dedifferentiate back to a quiescent form, in response to steroid and peptide hormones (reviewed by ). Milk provides an energy source, proteins and essential nutrients for the neonate, one of the key components of which is calcium (Ca2+). The rapid growth of the neonate, particularly the calcification of bones and teeth, places a high demand for Ca2+ in milk. Depending on the species, the concentration of total Ca2+ in milk ranges from 8 to 60 mM , a level well above the maternal blood level of total Ca2+. The secretory pathway and the apical plasma membrane play important roles in the transport of Ca2+ into milk [2–4].
Despite the importance of Ca2+ enrichment of milk, only recently have the Ca2+ transporters responsible for the accumulation of Ca2+ into milk begun to be identified. The best-characterized protein involved in the enrichment of milk with Ca2+ is the plasma membrane Ca2+ ATPase isoform 2 (PMCA2). This calcium efflux pump has a very restricted tissue expression and is present in specific parts of the brain and the inner ear [5–8]. PMCA2 is markedly up regulated during lactation, particularly splice variant PMCA2bw [5–7, 9], which localizes to the apical membrane of secretory cells [10, 11]. PMCA2 null mice show a 60% reduction in milk Ca2+ content, providing direct evidence for the role of PMCA2 in the apical transport of Ca2+ into milk during lactation .
The sequestration of Ca2+ into the secretory pathway during lactation appears to occur via the Golgi localized pump - secretory pathway Ca2+-ATPase isoform 2 (SPCA2). Like PMCA2, SPCA2 has a restricted tissue distribution  and is significantly up regulated during lactation . SPCA2 may also have a dual role in lactation due to its Mn2+ pumping ability . Both Ca2+ and Mn2+ are essential for enzymes necessary for the correct post-translational modification of milk proteins and lactose production .
Several different Ca2+ permeable ion channels are proposed as the mechanism by which Ca2+ enters the mammary epithelial cell from the maternal blood supply during lactation. Calcium channels suggested as involved in this pathway include TRPV5 and TRPV6 [17, 18]. However, recent studies suggest that the Orai1 calcium channel may be responsible for calcium influx into the mammary epithelial cell during lactation, since Orai1 mRNA levels increase in the mouse mammary gland during lactation . Indeed, Orai1 is at the basolateral membrane in mammary epithelial cells . ORAI1 is the canonical mechanism for store operated calcium entry (SOCE). SOCE is the activation of calcium influx into the cell upon the depletion of intracellular stores of Ca2+. Such a mechanism could be a powerful feedback loop to balance demand (the transport of Ca2+ into milk) with supply (the influx of Ca2+ into the mammary gland epithelial cell). Endoplasmic Ca2+ store level depletion is detected by STIM proteins; upon Endoplasmic Reticulum (ER) Ca2+ depletion, STIM proteins oligomerize and localize to ER-plasma membrane positions where they activate ORAI channels and promote SOCE [21–26]. The Orai1 isoform of ORAI channels is up regulated in mammary gland tissue samples taken from mice at lactation . However, levels of the canonical Orai1 activator Stim1 decline during lactation. The related isoform Stim2 is suggested as the possible mechanism of activation of Orai1 during lactation as this isoform does not decrease during lactation and is linked to the regulation of basal Ca2+ influx . Studies identifying the carboxyl terminal of SPCA2 as an activator of ORAI1 and the interaction between SPCA2 and ORAI1 in MCF-7 breast cancer cells, suggests that the up regulation of SPCA2 may not only serve to promote Ca2+ secretion during lactation, but also as an activator of Ca2+ influx. However, this has not been assessed in models of lactation. Another aspect of SPCA2 that has not yet been assessed in lactation is the role of the transcription factor MIST1, which is important in mammary gland development . A novel, truncated form of Spca2 was identified in Mist1−/− mice, but the presence of this form of Spca2 and its potential role in the regulation of Ca2+ transport during lactation has not been assessed. In these studies we used the HC11 model to further explore Ca2+ influx in mammary gland epithelial cells and to define the potential role of MIST1 regulation of Spca2 splicing during mammary gland development.
Basal Ca2+ influx is increased in differentiated HC11 cells
Calcium assays were conducted to assess changes in basal Ca2+ influx in response to differentiation of HC11 cells. Basal Ca2+ influx (assessed by the rate of Ca2+ increase upon addition of extracellular Ca2+) significantly increased in differentiated cells (Figure 1B and C) indicating that an increase in basal Ca2+ influx accompanies β-casein induction and may be a characterizing feature of the changes associated with lactation. We then sought to determine the role of Orai1 in this enhancement of Ca2+ influx.
Enhanced basal Ca2+ influx in differentiated HC11 cells is abolished by siRNA for Orai1
Spca2 in HC11 cells and mouse mammary tissue samples
β-Casein levels are unchanged with Orai1 silencing
Orai1 mRNA levels do not change with differentiation
The enrichment of milk with calcium is essential for the growing neonate. Our understanding of the specific calcium channels and pumps that are involved in this process is gradually evolving. However, the area that is least understood in the transport of Ca2+ into milk is the mechanism by which Ca2+ flows from the maternal blood supply into the mammary epithelial cell. In vivo studies implicate store operated Ca2+ entry as the calcium influx pathway involved, with Orai1 mRNA levels increasing in mouse mammary glands during lactation . Although Orai1 silencing reduces Ca2+ influx in human breast cancer cell lines , no studies have directly assessed calcium influx mediated by this pathway in in vitro models of lactation. The possible roles of the Orai1 activators Stim1, Stim2 and Spca2 have also not been assessed through direct assessment of Ca2+ influx in mammary gland epithelial cells. These questions were addressed in this study.
HC11 cells are a mouse mammary epithelial cell line derived from COMMA-1D cells, isolated from mid-pregnant Balb/c mice mammary glands [30, 31]. Unlike many other mammary cell models, HC11 cells do not need to be co-cultured with other cell types or with extracellular matrix proteins to help induce lactogenic differentiation [30, 32–34], making it suitable for the calcium influx assays used in this study. The expression of β-casein is essential for Ca2+ accumulation into milk, as a significant amount of calcium is present in casein micelles [2, 35]. Moreover, β-casein is a marker of differentiation in HC11 cells [36, 37].
Differentiation of HC11 cells with lactogenic hormones resulted in a statistically significant increase in basal Ca2+ influx compared to undifferentiated controls. This result is consistent with the hypothesis that lactation is associated with the remodelling of mammary gland epithelial cells to a more Ca2+ permeable phenotype associated with elevated basal Ca2+ influx. This increase in Ca2+ influx is likely to be required to meet the demands of the secretion and efflux of Ca2+ from the mammary gland epithelial cell into milk.
Silencing of Orai1 demonstrated that this augmented Ca2+ influx is via Orai1, since the difference in Ca2+ influx between differentiated and undifferentiated HC11 cells was abolished with the silencing of this calcium channel. Hence, consistent with elevated Orai1 mRNA levels during lactation, Orai1 appears to be a major contributor of the enhanced basal Ca2+ influx in mammary gland epithelial cells from a lactating host. Orai1 along with its activator Stim2 are regulators of basal Ca2+ levels . Our studies now suggest that this basal influx pathway is dynamic and may be up regulated during lactation. Future studies should assess how reported regulators of calcium transport during lactation, such as the calcium-sensing receptor, affect Orai1-mediated calcium influx in this and other models ).
McAndrews et al. compared 2 day (undifferentiated) and 8 day (with lactogenic hormones – differentiated) HC11 cultures and found elevated Orai1 mRNA in HC11 cells at 8 days of differentiation . These studies comparing cultures at day 8 with and without lactogenic hormones, suggest that it may be days in culture and/or confluence, which produces an increase in Orai1 mRNA levels. Lactogenic hormones may be responsible for the enhancement of Orai1 basal activity in HC11 cells. Orai1 does not appear to be a key pathway in the differentiation of HC11 cells given that Orai1 silencing had no effect on β-casein levels, this is in contrast to SCp2 cells where mammosphere formation was abolished with shOrai1 treatment . The lack of effects of Orai1 silencing on β-casein levels in HC11 cells indicates that the decrease in basal Ca2+ influx observed when Orai1 was silenced was not simply a consequence of the inhibition of HC11 differentiation.
ORAI1 is activated via a number of different mechanisms including the canonical ORAI1 activator and calcium store sensor STIM1, its related isoform STIM2, and SPCA2. STIM2 is proposed as an ORAI1 activator in lactation due to its important role in basal influx in HeLa, HUVEC and HEK293T cells . The maintenance of Stim2 mRNA levels during lactation  and reduction in basal [Ca2+]CYT in differentiated HC11 cells with Stim2 silencing also support this role. Other mechanisms of ORAI1 activation during lactation have also been proposed, such as calcium store independent activation by SPCA2 . Specific domains of SPCA2 protein activate ORAI1 , and Spca2 mRNA levels are pronouncedly increased during lactation . Activation of ORAI1 by SPCA2 may allow the demand for Ca2+ sequestration via this secretory pathway Ca2+ pump to promote the supply of Ca2+ through augmentation of Ca2+ influx. However, in these studies, and in contrast to Orai1 silencing, neither Stim1 nor Spca2 silencing abolished the augmentation of Ca2+ influx induced by differentiation of HC11 mammary epithelial cells. It could be that some in vitro systems have calcium influx pathways that are more sensitive to spca2 dependent modulation of Orai1, such has recently been reported in Scp2 cells . The ability of Stim2 silencing to abolish the enhanced Ca2+ influx associated with differentiation of HC11 cells, supports previous suggestions that Stim2 is a modulator of Orai1 during lactation, however, the inability of Stim2 silencing to replicate the magnitude of Orai1 silencing suggest other Orai1 activation mechanisms. Compensatory mechanisms amongst the Orai1 activators Stim1, Stim2 and Spca2 in this model and/or other factors such as the absence of coordinated polarization of mammary gland epithelial cell may be responsible for the results reported here. Further studies using 3D culture models in multiple mammary cell lines models, such as those recently published by Cross et al. in Spc2 cells , and in vivo studies with knockout animals are required to ultimately define the specific roles of Stim1, Stim2 and Spca2 and the activation of Orai1 in lactation.
The influx of Ca2+ across mammary gland epithelial cells is a key step in the supply of Ca2+ to the growing neonate during lactation. These studies using HC11 mammary gland epithelial cells are consistent with recent in vivo studies of mRNA levels during mammary gland development suggesting that Orai1 is an important pathway of Ca2+ influx during lactation.
Mouse mammary epithelial cells (HC11) were propagated in maintenance media containing Roswell Park Memorial Institute (RPMI)-1640 Medium (R8757, Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) and 10 mg/mL bovine insulin, as previously described . Cells were maintained in a 37°C, humidified 5% CO2, 95% air incubator. Cell lines were routinely tested for mycoplasma contamination.
HC11 cell differentiation and cell culture treatments
HC11 cells were seeded at 3500 cells/well into 96 well plates in maintenance media containing 10 ng/mL murine EGF. Fresh maintenance media with EGF was added at 24 h and siRNA treatment performed at 48 h in the presence of EGF and insulin. Following 24 h siRNA treatment media was replaced with maintenance media for 24 h. After 48 h siRNA treatment, media was replaced with either maintenance media supplemented with 1 μM dexamethasone and 5 μg/mL ovine prolactin (L6520, Sigma Aldrich) to differentiate the cells, or with maintenance media for proliferating non-differentiated cells. Fresh maintenance media was added at 24 h with or without dexamethasone and prolactin, as appropriate. Calcium assays were performed 144 h post plating. RNA was isolated from cells after calcium measurements were taken. Experiments were conducted in triplicate wells and all experiments were performed independently on four occasions.
siRNA transient transfection
siRNA transfection was performed using Dharmacon ON-TARGETplus SMARTpool™ siRNA (100 nM), comprising a pool of four siRNA sequences rationally designed with dual strand modification and use of an algorithm to reduce seed region matches. DharmaFECT1 transfection reagent (catalogue number T-2001-01) was used (0.4 μL/well) as per the manufacturer’s instructions. The following Dharmacon On-TARGETplus SMARTpool™ mouse siRNAs were used in this study: non-targeting (D-001810-10-05), Orai1 (L-056431-02-005), Stim1 (L-062376-00-0005), Stim2 (L-055069-01-0005) and Spca2 (L-065820-01-005). siRNA knockdown was checked using real-time RT-PCR for each experimental plate 24 h post treatment (Additional file 1).
Calcium assays were performed using a fluorometric imaging plate reader (FLIPRTETRA, Molecular Devices Corporation) using the no-wash cytosolic free calcium PBX Ca2+ Assay Kit (BD Biosciences) as previously described . Cells were seeded in 96 well black-walled plates (Corning). Intracellular Ca2+ measurements were performed with an excitation intensity of 470–495 nm and a 515–575 nm emission filter. Fluorescent values were normalized to the starting fluorescence and were expressed as relative [Ca2+]CYT. The slope of the curve was calculated based on points at 0 to 5 s post Ca2+ addition and were expressed as a percentage relative to the average readings for differentiated cells treated with non-targeting siRNA with maximum Orai1-mediated Ca2+ influx induced by 10 μM cyclopiazonic acid (CPA).
Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) as per the manufacturer’s instructions, and mRNA was quantitated using real time RT-PCR and a 7500 real time PCR system (Applied Biosystems). Mouse mammary tissues were obtained as previously described . Mouse β-casein (Mm00839664_m1), Orai1 (Mm00774349_m1), Spca2 (Mm01242899_m1), Spca2 exon 15–16 (Mm01242904_m1), Spca2 exon 26–27 (Mm01242916_m1), Mist1 (Mm00487695_m1), Stim1 (Mm00486423_m1), and Stim2 (Mm01223102_m1) were amplified using the TaqMan® gene expression assays, and the data normalized to Ppib (Mm00478295_m1) and Actb (Mm01205647_g1). Data were analyzed using the comparative Ct method as described previously . All data were normalized to proliferating HC11 cells treated with non-targeting siRNA. Data are shown as mean plus SD (n = 4).
All experimental treatments were conducted as three wells per experimental plate in quadruplicate. Results for the slope are presented as standard error of the mean (SEM) and statistical comparisons were performed using a two-way RM ANOVA matching by rows with a Bonferroni multiple comparison post-test. Real-time RT-PCR results are presented as standard deviation (SD) and were analyzed for significance using a paired t-test. All statistical analyzes were conducted using Prism Graph Pad, Version 5.04, Berkeley, CA, and significance was demonstrated at P < 0.05 where appropriate.
We would like to thank Prof. Melissa Brown from the University of Queensland, School of Chemistry and Molecular Biosciences, for providing the mammary tissue samples used in the project. This work was supported by National Health and Medical Research Council Grant 631347.
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