Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.


Introduction
Calcium-sensing receptor (CaSR), a cell-surface protein, is highly expressed in tissues and cells involved in systemic calcium homeostasis, including the parathyroid gland, kidney, and bone, where it contributes to the maintenance of systemic calcium within a narrow physiological window [1]. However, CaSR is also expressed in many other tissues and cells that are not primarily involved in extracellular calcium homeostasis, such as in the brain, skin, lungs, suggesting that this receptor plays additional physiological roles in the regulation of cell functions, such as cellular proliferation [2], differentiation [3] and apoptosis [4]. In the kidney, CaSR is well known to regulate calcium excretion and absorption in the renal tubules [5]. Interestingly, recent evidence indicates that CaSR is also expressed in glomeruli, and pharmacological activation of CaSR by the calcimimetic R-568 exerts a direct nephroprotective effect at the glomerular podocyte level [6,7]. A previous study showed that CaSR was expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induced cell proliferation [8]. However, nothing is currently known about the signaling cascades initiated by CaSR activation in MCs.
Although downstream effects can be highly varied, the first reactions following CaSR activation are common; stimulation of CaSR evokes an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [9]. CaSR belongs to family C of the G protein-coupled receptor superfamily. Stimulation of CaSR by an increase in extracellular Ca 2+ concentration ([Ca 2+ ] o ) or a polyamine (such as spermine) activates phospholipase C (PLC), which converts phosphatidylinositol 4,5-bisphosphate into inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 triggers Ca 2+ release from internal stores, resulting in an increase in [Ca 2+ ] i . However, the concomitant store depletion might mediate store-operated calcium entry (SOCE) through store-operated channels (SOCs) in the plasma membrane. Moreover, DAG can cause receptoroperated calcium entry (ROCE) by activating receptor-operated channels (ROCs). IP 3 -mediated Ca 2+ release, SOCE and ROCE all likely contribute to the increase in [Ca 2+ ] i upon activation of CaSR. IP 3 -mediated Ca 2+ release in response to CaSR stimulation has been widely investigated in many cell types; however, relatively little is known about calcium entry mechanism upon CaSR activation. SOCs and, in many cases ROCs, have been identified as canonical transient receptor potential (TRPC) channels. Furthermore, several studies indicated that TRPC channels are involved in the CaSR stimulation-induced calcium influx in some cell types, such as salivary ductal cells [10], MCF-7 breast cancer cells [2], aortic smooth muscle cells [11], keratinocytes [12], pulmonary neuroendocrine cells [13] and osteoclasts [14].
Studies from our laboratory and other laboratories have demonstrated that human MCs express TRPC channel proteins, including isoforms of TRPC1, 3, 4, and 6 [15,16]. In the present study, we investigated the role of TRPC channels in the CaSR activation-induced calcium influx and subsequent cell proliferation in human MCs. We determined that CaSR activation mediated TRPC3-and TRPC6-dependent calcium entry in a storeindependent manner. Furthermore, knockdown or pharmacological blockage of TRPC3 and TRPC6 inhibited the CaSR agonistinduced cell proliferation.

Cell culture and transfection
An stable human mesangial cell line (kindly donated by Dr. J. D. Sraer, Hopital Tenon, Paris, France) was established by transfection and immortalization by the viral oncogene large T-SV40 of human mesangial cells isolated from normal human glomeruli [17], and were cultured as described previously [16]. Briefly, the cells were cultured in RPMI1640 medium (HyClone, USA) containing 1 mM Ca 2+ supplemented with 10% fetal bovine serum (HyClone, USA) in 5% CO 2 at 37uC. Human MCs between passages 3 and 15 were used. A human breast cancer cell line MCF-7 was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), maintained in 5% CO 2 at 37uC in DMEM medium (HyClone, USA) containing 10% fetal bovine serum (HyClone, USA). Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA (Santa Cruz, USA) using the Xtreme GENE siRNA transfection reagent (Roche, Germany) according to the manufacturer's instructions. The transfected cells were assayed 24 to 48 h post-transfection.

Reverse transcription PCR and quantitative real-time PCR
Reverse transcription was performed using an RT system (Eppendorf Mastercycler, Hamburg, Germany) and a 10 ml reaction mixture. A High Capacity cDNA RT Kit (ABI Applied Biosystems, USA) was used for the initiation of cDNA synthesis .  The primer sequences used to amplify CaSR, TRPC1, TRPC3,  TRPC4 and TRPC6 were as follows (59-39):  CaSR sense CGGGGTACCTTAAGCACCTACGGCATC-TAA,  and  antisense  GCTCTAGAGTTAACGCGATCC-CAAAGGGCTC;  TRPC1 sense CGCCGAACGAGGTGAT,  and antisense GCACGCCAGCAAGAAA;  TRPC3 sense CGGCAACATCCCAGTG,  and antisense CGTAGAAGTCGTCGTCCTG;  TRPC4 sense CTCTGGTTGTTCTACTCAACATG,  and antisense CCTGTTGACGAGCAACTTCTTCT;  TRPC6 sense GCCAATGAGCATCTGGAAAT,  and antisense TGGAGTCACATCATGGGAGA. PCR cycling conditions for CaSR included one cycle of 10 min at 95uC, 35 cycles of 30 s at 95uC, 30 s at 55uC and 60 s at 72uC, and one cycle of 10 min at 72uC. The PCR products of CaSR were then separated on a 1% agarose gel and stained with ethidium bromide. Reverse transcriptase was omitted as a negative control for the RT-PCR to eliminate amplification from contaminating genomic DNA. All real-time PCR experiments were performed with SYBR Green PCR MasterMix (ABI Applied Biosystems, UK) using an ABI PRISM 7500 (ABI Applied Biosystems, USA). GAPDH was used as the internal control, and DDCt was calculated for each sample with the expression levels indicated by values of 2 2DDCt .

Western blot
Western blot was performed using a standard protocol. Human MCs were starved for 24 h in a serum-free medium prior to stimulation with high [Ca 2+ ] o . At the end of the 24 h incubation, the cells were harvested for western blot analysis. Anti-CaSR antibody (Affinity BioReagents, USA), anti-TRPC1, -TRPC3, -TRPC4, or -TRPC6 antibodies (Alomone Labs, Israel) or an antiactin antibody (Santa Cruz, USA) were used as primary antibodies. Fluorescence-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies (Invitrogen, USA) were used as secondary antibodies. Western blot bands were quantified using the Odyssey infrared imaging system (LI-COR Bioscience, USA).

Immunofluorescence
Immunofluorescence staining was performed on cultured MCs growing on coverslips using a standard protocol. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.4% Triton X-100 in PBS for 60 min at room temperature. The nonspecific binding sites were blocked with 50% goat serum in PBS for 60 min at 37uC. The cells were then incubated overnight at 4uC with anti-CaSR antibody (Affinity BioReagents, USA). After washing, cells were stained with Alexa Fluor 594 conjugated to goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) for 60 min at room temperature. Secondary antibody without prior antibody treatment was also included as negative controls. Cells were then stained with DAPI (Sigma, USA) for 15 min at room temperature to detect nuclei. After washing, samples were examined under a laser scanning confocal microscope (FV300; Olympus, Japan). Calibrations were performed immediately following each experiment. More than 50 cells were inspected per experiment, and photos of cells with typical morphology and staining are presented.

Fluorescence measurement of [Ca 2+ ] i
MCs were grown on coverslips and loaded in 1% physiological saline solution containing Pluronic F-127 (0.03%, Sigma, USA) and Fluo-3/AM (3 mM, Molecular Probes, USA) at 37uC for 45 min. After washing, the coverslips with cells were placed in a chamber containing HEPES-buffered Na + medium (HBM) that consists of the following (in mM): 137 NaCl, 5 KCl, 1 CaCl 2 , 1.2 MgCl 2, 0.44 KH 2 PO4, 4.2 NaHCO 3 , 10 glucose, and 20 HEPES; the pH was adjusted to 7.4 with NaOH. For the Ca 2+ -free HBM, Ca 2+ was omitted. MCs were then stimulated with a variety of agonists or inhibitors as described in the results, including spermine, NPS2390, U73122, thapsigargin (TG), SKF96365, 2aminoethoxydiphenyl borate (2APB), efonidipine, 1-oleoyl-2-acetyl-sn-glycerol (OAG) (all from Sigma Chemical Co., USA), and SN-6 (Tocris Bioscience, Bristol, UK). The fluorescence intensity of Fluo-3 in the cells was recorded by a laserconfocal scanning microscope (FV300; Olympus, Japan). The [Ca 2+ ] i was expressed as a pseudo-ratio value of the actual fluorescence intensity divided by the average baseline fluorescence intensity. Calibrations were performed immediately following each experiment. Data from 20 to 40 cells were summarized in a single run, and at least three independent experiments were conducted.

Cell proliferation assay
Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit (Roche, Germany) according to the manufacturer's protocol. Cells were seeded in a 96-well plate (5000 cells/well) and  The absorbance values correlate directly to the amount of DNA synthesis and therefore to the number of proliferating cells in culture. Stimulation is expressed as fold proliferation over basal growth of the control set as unity.

Statistical analysis
Data are presented as the means 6 SEMs with the indicated number (n) of experiments. Statistical analyses were performed using an unpaired t-test (SPSS 16.0), and graphs were plotted in GraphPad Prism 5 (GraphPad Software, Inc.). P,0.05 was considered statistically significant.

CaSR is expressed in human MCs.
To determine whether CaSR mRNA is expressed in cultured human MCs, RT-PCR was performed using specific primers for CaSR. As shown in Fig. 1A, a PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase, no PCR-amplified products were detected, indicating that the tested RNA samples were free of genomic contamination. As a positive control, an RT-PCR product from MCF-7 cells revealed a band of the same size as the human MCs. The expression of CaSR protein in human MCs was explored by Western blot analysis and immunostaining. As shown in Fig. 1B, a 130 kDa band, corresponding with the mature CaSR, was found in both human   MCs and in the positive control of MCF-7 cells. Immunostaining showed that CaSR protein was mainly localized at the plasma membrane along with some cytoplasmic localization (Fig. 1C). Taken together, these data demonstrate that CaSR is present in cultured human MCs.

Activation of CaSR stimulates an increase [Ca 2+ ] i in human MCs
To evaluate if the expression of CaSR protein is associated with the presence of functional receptors, Fluo-3/AM-loaded human MCs were stimulated by known CaSR agonists. As shown in Fig. 2A (Fig. 2C), indicating that the observed effect of CaSR activation was not agonistspecific. Additionally, the increase in [Ca 2+ ] i induced by spermine was dose-dependent (Fig. 2D) Fig. 2G and 2H, respectively. Taken together, these data confirm that CaSR protein is functionally expressed in human MC and activates a PLC-dependent [Ca 2+ ] i increase.

CaSR activation induces both intracellular Ca 2+ release and TRPC-dependent Ca 2+ influx
Because Ca 2+ mobilization from intracellular stores by CaSR agonists has been shown in many cell types, we investigated whether similar effects of CaSR agonists occur in human MCs. Cells were stimulated by spermine in the absence of extracellular Ca 2+ . As shown in Fig. 3A, 3 mM spermine induced an increase in [Ca 2+ ] i in Ca 2+ -free solutions. Accordingly, no Ca 2+ signal was ever observed after store depletion by 1 mM thapsigargin (TG), an endoplasmic reticulum Ca 2+ -ATPase inhibitor, further indicating that CaSR agonists stimulate Ca 2+ release from intracellular stores (Fig. 3B). Interestingly, as shown in Fig. 3A, the spermine-induced [Ca 2+ ] i increase in the absence of extracellular Ca 2+ was smaller than that observed in the presence of 1 mM [Ca 2+ ] o and had no Because TPRC channels, voltage-gated calcium channels and Na + /Ca 2+ exchangers are the main pathways for Ca 2+ influx in MCs [18], we examined the role of these pathways in CaSR agonist-induced [Ca 2+ ] i increase. As shown in Fig. 4A and 4B, pretreatment with SKF96365 (50 mM) or 2-APB (100 mM), nonselective TRPC channel blockers [19], significantly inhibited the 5 mM [Ca 2+ ] o -and 3 mM spermine-induced [Ca 2+ ] i increase, whereas pretreatment with efonidipine (10 mM, a voltage-gated calcium blocker) or SN-6 (10 mM, an specific inhibitor of NCX) had no apparent effect (Fig. 4C and 4D) Fig. 5B). However, this treatment did not lead to increases in TRPC1 or TRPC4 expression (p.0.05; n = 3; Fig. 5).

TRPC3 and TPRC6 are required for the CaSR agonistinduced [Ca 2+ ] i increase
To investigate whether TRPC3 and TRPC6 are involved in [Ca 2+ ] i increase induced by CaSR activation, we used siRNA technology to downregulate TRPC3 and TRPC6 expression in human MCs. The specificity and efficiency of TRPC3-siRNA and TRPC6-siRNA was confirmed by real-time RT-PCR and Western blot analyses, indicating that this procedure decreased the expression level of endogenous TRPC3 and TRPC6 without affecting other TRPC channels (Fig. 6A-D). Compared with cells transfected with scrambled siRNA, transfection with TRPC3 siRNA and TRPC6 siRNA partially, but significantly, inhibited the spermine-induced [Ca 2+ ] i increase by 66.47% and 63.20%, respectively (p,0.05, n = 3; Fig. 7A  The release from calcium stores is not essential for TRPC3-and TRPC6-mediated calcium influx by CaSR activation TRPC3 and TRPC6, as receptor-operated channels, can be activated by DAG and mediate ROCE in a variety of cell types. Therefore, we tested whether TRPC3 and TRPC6 can be activated by OAG, a membrane-permeable DAG analogue, in human MCs. As expected, the OAG-induced Ca 2+ influx was significantly reduced by transfection with TRPC3 siRNA and TRPC6 siRNA compared with transfection with scrambled siRNA (Fig. 8A-C). Importantly, OAG significantly enhanced the [Ca 2+ ] o -and spermine-induced [Ca 2+ ] i increases ( Fig. 8D and  8E), suggesting that CaSR agonists likely evoke calcium entry via receptor-operated channels.
To further demonstrate that calcium influx stimulated by CaSR agonists does not require the release of calcium stores, we depleted stores with TG before CaSR stimulation. TG blocks Ca 2+ -ATPase located in the membrane of the endoplasmic reticulum (ER) and other intracellular vesicular store compartments. As shown in Fig. 9A, in the presence of 1 mM TG, a restoration of extracellular calcium from 0 to 0.5 mM induced an expected rise in [Ca 2+ ] i , which was due to SOCE, and 3 mM spermine evoked an additional substantial increase in [Ca 2+ ] i under conditions where Ca 2+ stores were already depleted. However, the additional substantial increase in [Ca 2+ ] i was blocked by NSP2390, a CaSR antagonist. Similar results were obtained with a 5 mM [Ca 2+ ] oinduced calcium influx (Fig. 9B). These results suggest that TRPC3-and TRPC6-mediated calcium influx by CaSR activation does not require the release of calcium stores.  (Fig. 10C). Furthermore, transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuated the promotion of proliferation by [Ca 2+ ] o , respectively, compared with scramble RNA (Fig. 10D). Taken together, these data indicate that TRPC3 and TRPC6 play a role in cell proliferation induced by CaSR stimulation.

Discussion
CaSR has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions [1]. A previous study showed that CaSR is localized not only in the renal tubules but also in the glomeruli [20]. Further work indicated that CaSR is functionally expressed in mouse mesangial cells and modulates cell proliferation [8]. Unfortunately, no reports have been made on the mechanism by which CaSR induces calcium-dependent signaling in MCs. In this study, we demonstrate that CaSR is functionally expressed in a human MC cell line. Further, our data reveal that CaSR activation induces an increase in [Ca 2+ ] i via both calcium entry by TRPC3 and TRPC6 and release from intracellular stores. Furthermore, TRPC3 and TRPC6 are associated with CaSR activation-induced cell proliferation in human MCs.
CaSR can be activated by two types of agonists. Type I agonists are divalent cations, such as Ca 2+ and Mg 2+ , which directly activate the receptor. As shown in this study, the EC 50 of Ca 2+ in human MCs is 4.93 mM, close to that of HEK-293 cells transfected with the human CaSR (4.1 mM) [21]. The type II agonists, such as spermine, amino acids, and ionic strength, are better referred to as modulatory substances, which allosterically increase the calcium affinity of the receptor [9]. The highly cooperative process of Ca 2+ binding to CaSR allows CaSR to function as a sensitive detector of Ca 2+ , thereby quite easily distinguishing small (,200 mM) fluctuations in the [Ca 2+ ] o [9]. Moreover, spermine is a uremia toxin that has been implicated as a potential mediator of chronic kidney disease-associated clinical abnormalities [22]. Therefore, CaSR could play an important role in MCs under physiological and pathophysiological conditions, although the typical physiological [Ca 2+ ] o is approximately 1.3 mM [23], much lower than that required CaSR activation in MCs as determined from our data.
Generally, exposure of cells to CaSR agonists commonly elicits a [Ca 2+ ] i increase through interactions between CaSR and PLC, which are mediated by the G q/11 subunits of heterotrimeric G proteins [9,23] ] i increase is composed of an initial rapid increase and followed by a sustained increase, consistent with a previous study in mouse MCs [8]. The overshooting peaks may represent intracellular Ca 2+ mobilization, and the sustained elevation may represent activation of Ca 2+ influx. Indeed, our data indicate that CaSR stimulation induces TRPC-dependent calcium entry as well as calcium release from intracellular stores. Further, our results suggest that TRPC3 and TRPC6 may be responsible for the CaSR activation-induced calcium influx because of the following: (i) stimulation of CaSR by high [Ca 2+ ] o enhanced the expression of TRPC3 and TRPC6, rather than of TRPC1 and TRPC4, and (ii) siRNA targeting of TRPC3 and TRPC6 attenuated the CaSR activation-induced calcium influx. Previous patch clamp experiments revealed that stimulation of CaSR induces TRPC-like nonselective cation currents in HEK293 cells stably transfected with CaSR [24] and in MCF-7 breast cancer cells [25]. Moreover, several isoforms of TRPC channels, dependent on the cell type, have been implicated in the CaSR activation-induced Ca 2+ entry, such as TRPC3 in salivary ductal cells [10], TRPC1 in MCF-7 breast cancer cells [2] and keratinocytes [12], and TRPC6 in aortic smooth muscle cells [11] and cardiac myocytes [4]. These studies support the contribution of TRPC channels in the CaSR activation-induced Ca 2+ entry.
In MCs, as in other cell types, TRPC3 channels and TRPC6 channels are considered to be ROCs [16,19], whereas TRPC1 channels and TRPC4 channels are SOCs [26]. Given that a functional hallmark of ROCs is that they can be directly activated by DAG without depleting intracellular stores, CaSR agonists may be able to induce the activation of ROCs because PLC-mediated DAG production following CaSR stimulation has been demonstrated in a number of studies [1,9,27,28]. In this study, we show that OAG, a membrane-permeable DAG analogue, significantly enhances the CaSR agonist-induced [Ca 2+ ] i increase. Moreover, further experiments with TG to deplete intracellular stores before CaSR stimulation revealed that the effects of CaSR agonists on [Ca 2+ ] i still occurred, suggesting that calcium influx stimulated by CaSR agonists do not require depletion of intracellular stores. This idea is supported by the results obtained in osteoclasts and aortic smooth muscle cells, where CaSR activation mediates ROCE [11,14]. Furthermore, our findings is concordant with several observations that have shown that TRPC3-TRPC6 channels mediate store-independent Ca 2+ entry in prostate smooth muscle cells [29], MDCK [30], and cardiac myocytes [31]. However, in other cell types, such as MCF-7 breast cancer cells and keratinocytes, CaSR activation mediates SOCE [2,3], suggesting that CaSR stimulation mediates Ca 2+ entry in a cell-specific manner. In this study, we cannot exclude the possibility that SOCE occurs simultaneously with ROCE upon CaSR activation because CaSR stimulation induces Ca 2+ release from intracellular stores and can thereby directly or indirectly affect the TRPC3 and TRPC6 activities [19].
Finally, we determined the role of TRPC3 channels and TRPC6 channels in CaSR stimulation-induced cell proliferation, showing that both pharmacological blockage and siRNA knock down of TRPC3 and TRPC6 inhibited high [Ca 2+ ] o -induced mesangial cell proliferation. A similar role was described in MCF-7 breast cancer cells. However, whether the role of TRPC3 and TRPC6 in the CaSR stimulation-induced cell proliferation is mediated by changing intracellular calcium or results from multiple postreceptor responses has yet to be determined. Regardless, our study demonstrates that CaSR modulates extracellular calcium entry and proliferation via TRPC3/6 channels in human MCs.