Calcium-Sensing Receptor Stimulation Induces Nonselective Cation Channel Activation in Breast Cancer Cells
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- Hiani, Y.E., Ahidouch, A., Roudbaraki, M. et al. J Membrane Biol (2006) 211: 127. doi:10.1007/s00232-006-0017-2
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The calcium-sensing receptor (CaR) is expressed in epithelial ducts of both normal human breast and breast cancer tissue, as well as in the MCF-7 cell line as assessed by immunohistochemistry and Western blot analysis. However, to date, there are no data regarding the transduction pathways of CaR in breast cancer cells. In this study, we show that a CaR agonist, spermine, and increased extracellular Ca2+ ([Ca2+]o) sequentially activate two inward currents at –80 mV. The first was highly permeable to Ca2+ and inhibited by 2-aminophenyl borate (2-APB). In contrast, the second was more sensitive to Na+ and Li+ than to Ca2+ and insensitive to 2-APB. Furthermore, intracellular dialysis with high Mg2+, flufenamic acid or amiloride perfusion was without any effect on the second current. Both currents were inhibited by La3+. Calcium imaging recordings showed that both [Ca2+]o and spermine induced an increase in intracellular calcium ([Ca2+]i) and that removal of extracellular Ca2+ or perfusion of 2-APB caused a decline in [Ca2+]i. It is well known that stimulation of CaR by an increase in [Ca2+]o or with spermine is associated with activation of phospholipase C (PLC). Inhibition of PLC reduced the [Ca2+]o-stimulated [Ca2+]i increase. Lastly, reverse-transcriptase polymerase chain reaction showed that MCF-7 cells expressed canonical transient receptor potential (TRPCs) channels. Our results suggest that, in MCF-7 cells, CaR is functionally coupled to Ca2+-permeable cationic TRPCs, for which TRPC1 and TRPC6 are the most likely candidates for the highly selective Ca2+ current. Moreover, the pharmacology of the second Na+ current excludes the involvement of the more selective Na+ transient receptor potential melastatin (TRPM4 and TRPM5) and the classical epithelial Na+ channels.
KeywordsCalcium-sensing receptorCationic currentTransient receptor potential channelBreast cancer cell
Breast cancer is the most common cancer and a leading cause of cancer-associated death in women (Boring et al., 1994). It most commonly metastasizes to the bone, with 70% of patients dying of breast carcinoma having bone metastases (Mundy, 1997). Consequently, tumors induce bone resorption and lead to the release of large quantities of Ca2+ into the bone microenvironment. The local Ca2+ level at resorption sites has been reported to rise as high as 40 mM (Silver, Murrills & Etherington, 1988). It has been reported that high Ca2+ concentrations stimulate the calcium-sensing receptor (CaR), thereby inducing parathormone-related peptide (PTHrP) production by MCF-7 and MDA-MB-231 breast cancer cells (Sanders et al., 2000). High levels of PTHrP contribute to increased bone resorption and to the osteolysis observed in association with metastasis of epithelium-derived tumors to bone (Rodland, 2004). The potential role of CaR in modulating PTHrP secretion has been investigated in breast cancer (Sanders et al., 2000), prostate cancer (Sanders et al., 2001), glial tumors (Chattopadyay et al., 2000) and Leydig cell tumors (Buchs et al., 2000). Recently, Li, Huang & Peng (2005) reported overexpression of CaR in some cancer tissues and suggested that CaR may play a role in cancer progression.
CaR is a G protein-coupled receptor, originally cloned from bovine parathyroid cells (Brown et al., 1993), which plays an important role in the maintenance of systemic Ca2+ homeostasis. It has been reported that CaR stimulation by an increase in extracellular Ca2+ concentration ([Ca2+]o) or a polyamine (spermine) is associated with activation of phospholipase C (PLC) (Huang, Handlogten & Miller, 2002) and increases the levels of intracellular inositol 1,4,5-trisphosphate (IP3) (Guise et al., 1996). It is well known that PLC cleaves phosphatidylinositol 4,5-biphosphate into IP3 and diacylglycerol (DAG). IP3 releases Ca2+ from intracellular stores, and the concomitant store depletion activates store-operated Ca2+ channels (SOCs), which in many cases have been tentatively identified as transient receptor potential (TRP) family members (Zitt, Halaszovich & Luckhoff, 2002; Pedersen, Owsianik & Nilius, 2005; Parekh & Putney, 2005). Moreover, DAG is also capable of activating some other TRPs directly, without depleting intracellular Ca2+ stores (Pedersen et al., 2005).
In breast cells, CaR stimulation induces an increase in intracellular Ca2+ concentration ([Ca2+]i) (Parkash, Chaudhry & Rhoten, 2004) as well as PTHrP secretion (Sanders et al., 2000). Furthermore, a cationic nonselective current has been recorded both in response to CaR stimulation in hippocampal neurons (Ye et al., 1996a, 1997) and in HEK293 cells stably transfected with human CaR (Ye et al., 1996b). The greater incidence of this cationic current has been associated with U373 human astrocytoma cell proliferation (Chattopadhyay et al., 2000) and with differentiation of human promyelocytic leukemia cells (Yamaguchi et al., 2000). However, nothing is currently known about the pharmacological and electrophysiological properties of this cationic current and the link between the CaR and TRP channels (TRPCs).
We combined electrophysiological and molecular methods to demonstrate, for the first time, that both CaR agonists (spermine and Ca2+) sequentially activate two cationic currents: a primary highly Ca2+-sensitive current (I1), which is always followed by a secondary highly Na+-sensitive stronger one (I2). Furthermore, the pharmacological and electrophysiological properties of the primary current, on the one hand, and the transcripts of canonical TRPCs that we have found in MCF-7 cells, on the other hand, lead us to suggest that TRPC1 and/or TRPC6 might be candidates for the first current induced by the activation of CaR.
Materials and Methods
MCF-7 cells between passages 149 and 210 were cultured in Eagle’s minimum essential medium supplemented with 5% fetal calf serum, 2 mML-glutamine and 0.06% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and maintained at 37°C in a humid atmosphere of 5% CO2 in air.
For electrophysiological analysis, cells were cultured in 35-mm Petri dishes at a density of 5 × 104 for 2 days before patch-clamp experiments. Currents were recorded in voltage-clamp mode, using an Axopatch 200 B patch-clamp amplifier and a Digidata 1200 interface (both from Axon Instruments, Burlingame, CA). PClamp software (v. 6.03, Axon Instruments) was used to control voltage as well as to acquire and analyze data. The whole-cell mode of the patch-clamp technique was used with 3–5 MΩ resistance borosilicate fire-polished pipettes (Hirschmann® Laborgerate, Eberstadt, Germany). Seal resistance was typically in the 10–20 GΩ range. The maximum uncompensated series resistance was <10 MΩ during whole-cell recordings, so the voltage error was <5 mV for a current amplitude of 500 pA. Recordings where series resistance resulted in errors >5 mV in voltage commands were discarded. Whole-cell currents were allowed to stabilize for 5 min before being measured. Membrane capacitance was measured by voltage clamp with a voltage pulse after completion of a whole-cell patch-clamp procedure, and compensation of the electrode capacitance with electronic circuits was built into the patch-clamp amplifier. Results were expressed using current densities instead of current amplitude. The MCF-7 cell surface was thus estimated by measuring membrane capacitance (30 ± 1.7 pF, n = 45). Currents were recorded using the whole-cell patch-clamp technique during ramps from −80 to +80 mV, applied from a holding potential of −40 mV for 250 ms. The current value was measured at the end of the ramp protocol at −80 mV.
Cells were allowed to settle in Petri dishes placed at the opening of a 250-μm inner diameter capillary for extracellular perfusions. The cell under investigation was continuously superfused with control or test solutions. All electrophysiological experiments were performed at room temperature.
External and internal solutions had the following compositions (in mM): external, NaCl 100, KCl 5, MgCl2 1 and HEPES 10 at pH 7.4 (TEAOH); internal, CsCl 150, HEPES 10, ethyleneglycoltetraacetic acid (EGTA) 0.1 and MgCl2 2 at pH 7.2 (CsOH). Extracellular and intracellular osmolarity values measured with a freezing-point depression were 300 and 292 mOs, respectively. In order to completely block K+ channels, we added tetraethylammonium (TEA) at 20 mM to the extracellular medium. 2-Aminophenyl borate (2-APB), U73122 and U73343 (Sigma, Saint Quentin Fallavier, France) was made in dimethyl sulfoxide (DMSO). Final concentrations were obtained by appropriate dilution in an external control solution. The final concentration of DMSO was <0.1%. For the Na+-free solution, Na+ was replaced by choline and the pH was adjusted to 7.4 by TEAOH.
Results were expressed as mean ± standard error (SE). Experiments were repeated at least three times. Student’s t-test was used to compare treatment means with electrophysiological control means (paired t-test). P < 0.05 was considered significant.
MCF-7 cells were grown on glass coverslips for calcium imaging experiments. The cytosolic calcium concentration was measured using Fura-2-loaded cells. MCF-7 cells were loaded for 1 h at room temperature with 3.3 μM Fura-2/AM prepared in saline solution and subsequently washed three times with the same dye-free solution. The coverslip was then transferred into a perfusion chamber of a Zeiss (Thornwood, NY) microscope equipped for fluorescence. Fluorescence was excited at 350 and 380 nm alternately, using a monochromator (Polychrome IV; TILL Photonics, Planegg, Germany), and captured by a Cool SNAP HQ camera (Princeton Instruments, Evry, France) after filtration through a long-pass filter (510 nm). Metafluor software (v. 6.2r6; Universal Imaging, West Chester, PA) was used for acquisition and analysis. All recordings were carried out at room temperature. The cells were continuously perfused with the saline solution, and chemicals were added via the perfusion system. The flow rate of the whole-chamber perfusion system was set at 1 ml/min, and the chamber volume was 500 μl.
REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION ANALYSIS
Sequences of selected oligonucleotides used as RT-PCR primers
Position in GenBank sequence (accession number)
Expected fragment size (bp)
Forward: 5′- TTCCTCTCCATCCTCTTCCTCG-3′
458 for TRPC1,356 for TRPC1A,298 for TRPC1B
Reverse: 5′- CATAGTTGTTACGATGAGCAGC-3′
383 for TRPC3
781 for TRPC4
625 for TRPC6,
277 for TRPC6γ
477 for TRPC7
CAR STIMULATION INDUCES TWO CATIONIC CURRENTS IN MCF-7 CELLS
Because the local Ca2+ concentration near resorbing osteoclasts may rise as high as 40 mM (Silver et al., 1988), metastatic tumor cells in bone could be exposed to very high Ca2+ levels. In this study, we tested the effects of 10 and 20 mM [Ca2+]o on MCF-7 breast cancer cells, focusing on the regulation of calcium and sodium entry.
THE PRIMARY CURRENT MEASURED AT – 80 MV IS 2-APB-SENSITIVE AND CARRIED BY CA2+
Stimulation of CaR by both [Ca2+]o and spermine induced activation of PLC (Huang et al., 2002). This pathway eventually results in the activation of IP3 store-dependent and store-independent membrane channels, both of which are thought to belong to the TRPC family (Pedersen et al., 2005; Beech, 2005).
To further validate the involvement of TRPCs in the CaR stimulation of MCF-7 cells, we tested the ability of 2-APB to interfere with the effect of [Ca2+]o and spermine on Ca2+ homeostasis. An increase in [Ca2+]o from 2 to 20 mM elicited an increase in [Ca2+]i (n = 41, Fig. 2E). Perfusions of 2-APB at 50 μM reversibly blocked CaR-induced Ca2+ entry (n = 41, Fig. 2E). Moreover, removal of extracellular Ca2+ caused a sharp decline in [Ca2+]i (n = 41, Fig. 2E), suggesting that virtually most of the Ca2+ entered the cell from the extracellular space. Perfusion of 50 μM 2-APB caused a sharp and reversible decline of the 1 mM spermine-induced [Ca2+]i rise in MCF-7 cells (n = 54, Fig. 2F). The removal of extracellular Ca2+ induced a rapid decline in [Ca2+]i (n = 54, Fig. 2F).
INVOLVEMENT OF THE PLC PATHWAY IN [CA2+]O-INDUCED INTRACELLULAR CALCIUM INCREASE
DOSE DEPENDENCE OF [CA2+]I AND I1 AND I2 CURRENT DENSITIES ON [CA2+]O-INDUCED CAR STIMULATION
THE SECOND CURRENT IS NA+-AND LI+-SENSITIVE AND DEPENDENT ON THE PRIMARY ONE
In all our experiments, I2 always appeared after I1 activation, thus suggesting a dependent relationship between these two currents. To find out if this were true, we stimulated CaR in the presence of 50 μM 2-APB. Figure 6C shows that in the presence of 2-APB no currents were activated but, as soon as we washed the 2-APB out, we induced sequential activation of I1 followed by I2. Extracellular perfusion of 500 μM La3+ completely inhibited I2 (Fig. 6C). These results show that I2 is probably activated by [Ca2+]i. Similar results were obtained with La3+ (data not shown, n = 10).
PHARMACOLOGY OF I2
These results demonstrate that I2 could not be one of the TRPMs mentioned above. Moreover, as epithelial Na+ channels are mostly expressed in epithelial-type cells, we tested the effect of amiloride. Extracellular perfusion of amiloride 100 μM failed to inhibit I2 current (n = 8, Fig. 7C).
EXPRESSION OF PLC-COUPLED TRPCS IN MCF-7 CELLS
CaR is expressed in the epithelial ducts of the normal human breast (VanHouten, 2005; VanHouten et al., 2004; Cheng et al., 1998). It is localized in the laterobasal membrane and interacts with ionic transporters (VanHouten, 2005). Furthermore, CaR has been characterized both in breast cancer tissue and in breast MCF-7 and MDA-MB-231 cells (Cheng et al., 1998; Sanders et al., 2000). It has also been established that CaR can regulate the production of PHTrP, which enhances MCF-7 breast cancer cell proliferation, adhesion, migration and invasion via an intracrine pathway (Rodland, 2004; Falzon & Du, 2000; Shen, Qian & Falzon, 2004). In different types of cells, including MCF-7, CaR stimulation by [Ca2+]o or spermine elicits an increase in [Ca2+]i (Nemeth & Scarpa, 1987; Parkash et al., 2004). Furthermore, CaR stimulation is accompanied by PLC stimulation and IP3 production (Brown et al., 1987; Brown & MacLeod, 2001; Huang et al., 2002; Jiang et al., 2002). In this study, we present, for the first time, evidence of the role of CaR in Ca2+ signaling in MCF-7 cells. Furthermore, we show that the CaR-stimulated Ca2+ transmembrane influx was followed by an Na+ one and that activation of these currents may involve a store-dependent and/or independent mechanism.
Our results support a role of CaR in MCF-7 cells, an estrogen-positive human breast cancer cell line, in sensing and responding to change in [Ca2+]o (Figs. 2 and 3). A nonoscillatory response was observed in MCF-7 cells instead of the [Ca2+]i oscillations that were observed in HEK293 cells expressing CaR (Jiang et al., 2002). Moreover, our results are similar to those reported in MCF-7 by Parkash et al. (2004), who found a median effective concentration (EC50) of about 21 mM.
It has been reported that CaR activation by neomycin, spermine or elevated [Ca2+]o induces activation of nonselective cationic channels (Ye et al., 1996a, 1997; Chattopadhyay et al., 2000). More recently, Fatherazi et al. (2004) reported that in gingival cells CaR activation by 10 mM [Ca2+]o induced activation of two cationic currents (at -80 mV). Both currents are supported by Ca2+ influx. The present whole-cell patch-clamp results show that CaR activation sequentially induced two cationic currents, the first being highly Ca2+-sensitive followed by a second Na+ one. Our first current closely resembles the second current reported by Fatherazi et al. (2004). However, no Na+ current was recorded by Fatherazi et al. (2004). Our second current seems to be dependent on [Ca2+]i since it was always activated after the I1 current. This channel is nonselective since it conducts Li+, Na+ and Cs+. It was inhibited by neither high Mg2+, flufenamic acid nor spermine dialysis, thus suggesting that the channel is not a member of the reported TRPM 4, 5, 7 family. However, we do not exclude the involvement of other TRPM-like channels yet to be identified. Moreover, I2 was insensitive to amiloride (100 μM), excluding the involvement of Na+-epithelial channels. The increase in [Ca2+]i may also activate an Na+/Ca2+ exchanger, thereby inducing Na+ entry. However, the large amplitude of the second current excludes this hypothesis.
Our study provides direct evidence that CaR is functionally coupled to transmembrane Ca2+ entry via the PLC-catalyzed inositol phospholipid-breakdown signaling pathway, presumably through activation of channels of the TRP family. IP3 and DAG are two factors which are probably involved in Ca2+ entry in response to CaR stimulation by agonists. The first is related to the IP3-evoked depletion of intracellular Ca2+ stores and the subsequent activation of plasmalemmal SOCs, also thought to belong to the TRPC family (Pedersen et al., 2005; Parekh & Putney, 2005). The second, however, is related to direct DAG-mediated activation of other TRP members (Pedersen et al., 2005).
Our results show that MCF-7 cells pretreated with U73122 (a specific inhibitor of PLC) abolished the [Ca2+]o-stimulated [Ca2+]i increase, indicating that both store-dependent and independent TRPs are involved in the CaR response. Moreover, RT-PCR analysis showed the presence of the specific mRNA for only TRPC1 and TRPC6. The details of the molecular nature of the primary Ca2+-sensitive channel remain to be explored. However, a number of observations argue that the underlying cationic channels are closely related to SOCs. (1) 2-APB at low concentrations is capable of blocking nearly 100% of CaR-induced I1 and [Ca2+]I; (2) the CaR-induced I1 current is insensitive to RR, a potent inhibitor of such members of the transient receptor potential vanilloid (TRPV) family; and (3) spermine had no effect after treatment with thapsigargin, suggesting that there was no involvement of a TRP family member activated by DAG. Moreover, a correlation with the permeation and pharmacological profiles of the first cationic current, I1, in MCF-7 cells with those described for TRPC6 shows a number of notable differences: (1) I1 was highly permeable to Ca2+, in contrast to TRPC6 which is permeable to Ca2+ and Na+; (2) the increase in [Ca2+]o increased I1 amplitude, while it reduced TRPC6 activity; and (3) dialysis with 100 μM flufenamic acid, a TRPC6 activator, was without effect on I1. Taken together, these finding suggest that the CaR-activated cationic channels in MCF-7 cells are probably heterotetramultimers that necessarily include both TRPC6 and TRPC1 together with some other TRP members, which confer the property of SOC gating and, in combination, determine the resultant permeation and pharmacological characteristics of the whole channel.
Changes in [Ca2+]i are known to play an important role in the regulation of PTHrP secretion by MCF-7 cells (Brown et al., 1993). The MCF-7 cell line expresses functional PTH/PTHrP receptors, and PTHrP affects its growth in an autocrine/paracrine manner (Liapis et al., 1993). In this way, the CaR-induced TRP activation (store-dependent and/or independent) permits Ca2+ influx and an increase in [Ca2+]i. Moreover, the Na+ entry by a nonselective cation channel, which could be a member of the TRPM family, may induce an inverse function of the Na+/Ca2+ exchanger, thereby causing an additional Ca2+ entry, which allows a sustained increase in [Ca2+]i and the secretion of PTHrP and/or changes in gene expression, such as estrogen receptor downregulation in MCF-7 cells (Journe et al., 2004).
In conclusion, our findings provide new information about nonselective cationic channels supporting CaR-activated Ca2+ influx pathways in MCF-7 cells. These findings therefore contribute to the understanding of the functional significance of the presence of CaR in breast cancer cells.
We thank Jean François Lefebvre and Philippe Delcourt for their excellent technical assistance. This work was supported by the Ministère de l’Education Nationale, the Ligue Nationale Contre le Cancer, the Association pour la Recherche Contre le Cancer and the Region Picardie, France and by grants from Morocco.