TRPC channels are involved in calcium-dependent migration and proliferation in immortalized GnRH neurons

Cell Calcium 49 (2011) 387–394

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TRPC channels are involved in calcium-dependent migration and proliferation in immortalized GnRH neurons Paolo Ariano a,b,e , Simona Dalmazzo a,b , Grzegorz Owsianik c , Bernd Nilius c , Davide Lovisolo a,b,d,∗ a

Department of Animal and Human Biology, University of Torino, Italy NIS Centre of Excellence for Nanostructured Interfaces and Surfaces, University of Torino, Italy Department of Molecular Cell Biology, Laboratory of Ion Channel Research, Katholieke Universiteit Leuven, Leuven, Belgium d Neuroscience Institute of Torino, University of Torino, Italy e Center for Space Human Robotics, The Italian Institute of Technology, Torino, Italy b c

a r t i c l e

i n f o

Article history: Received 29 October 2010 Received in revised form 3 March 2011 Accepted 22 March 2011 Available online 20 April 2011 Keywords: GnRH GN11 Migration TRPC Calcium

a b s t r a c t Gonadotropin-releasing hormone (GnRH)-secreting neurons are key regulators of the reproductive behaviour in vertebrates. These neurons show a peculiar migratory pattern during embryonic development, and its perturbations have profound impact on fertility and other related functional aspects. Changes in the intracellular calcium concentration, [Ca2+ ]i , induced by different extracellular signals, play a central role in the control of neuronal migration, but the available knowledge regarding GnRH neurons is still limited. Our goal was to investigate mechanisms that may be involved in the Ca2+ dependence of the migratory behaviour in these neurons. We focused on the “classical” Transient Receptor Potential (TRPC) subfamily of Ca2+ -permeable cation channels, recently shown to be involved in other aspects of neuronal development. Using GN11 cells, immortalized early stage GnRH neurons, we set to investigate Ca2+ signals under basal conditions and in the presence of a well-established motogen, fetal calf serum (FCS), and the effect of pharmacological TRPC agonists and antagonists on Ca2+ oscillations, cell motility and proliferation. We have found that a subpopulation of GN11 cells shows spontaneous Ca2+ transients and that this activity is increased in the presence of serum. Quantitative real-time PCR showed that transcripts of some TRPC members are expressed in GN11 cells. Interestingly, pharmacological experiments with inhibitors, SKF-96365, lanthanum, anti-TRPC1 antibody, and activators, 1-oleil 2-acetyl-sn-glycerol, of TRPCs suggested that the activation of these channels can account for both the basal Ca2+ oscillations and the increased activity in the presence of FCS. Moreover, functional studies using the same pharmacological tools supported their involvement in the control of motility and proliferation. Thus, our data provide evidence for the involvement of Ca2+ -permeable channels of the TRPC subfamily in the control of functional properties of neurosecretory cells and neuronal motility. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Gonadotropin-releasing hormone (GnRH)-secreting neurons are key regulators of the reproductive behaviour in vertebrates [1,2]. The maturation of the GnRH system during embryonic development presents quite peculiar features. GnRH neurons originate in the olfactory placode and migrate to the hypothalamus [3–5] where they send axonal processes to portal vessels. Perturbations of the migratory process can lead to several pathologies and have a profound impact on fertility and other related functional aspects [1,6].

∗ Corresponding author at: Department of Animal and Human Biology, University of Torino, Via A. Albertina, 13, Torino, Italy. Tel.: +39 011 6704668. E-mail address: [email protected] (D. Lovisolo). 0143-4160/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2011.03.007

Fetal calf serum [7,8] and a variety of extracellular factors such as hepatocyte growth factor (HGF) [9,10], semaphorins [11,12], neurotransmitters and neuropeptides (see e.g., [13]) and cell adhesion molecules [1,14] have been shown to influence migration either of immature GnRH neurons (for a review see [15]) or of their immortalized counterpart, the GN11 cell line [16]. Up-to-date little is known about the intracellular mechanisms activated by these extracellular cues and their gradients. Calcium (Ca2+ ) is an ubiquitous second messenger that has achieved a well established role in the control of neuronal migration [17,18], and changes in its intracellular concentration, [Ca2+ ]i , can be induced by a wide set of extracellular signals. Regarding GnRH neurons, the available knowledge is still limited. In early stage GnRH neurons, it has been shown that migration depends on the presence of extracellular Ca2+ and only in part from Ntype voltage-dependent Ca2+ channels [19]. Moreover, it has been

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recently suggested that Ca2+ levels can affect migration of GN11 cells through the Ca2+ -sensitive phosphatase calcineurin [20]. In order to understand which mechanisms may be involved in the calcium dependence of the migratory behaviour in these neurons, we set to investigate, in GN11 cells, Ca2+ signals under basal conditions and in the presence of the well-established motogen, fetal calf serum (FCS), and the effects of manipulating the pathways responsible for these oscillations on motility and cell proliferation. To this purpose, we focussed on the “classical” Transient Receptor Potential (TRPC) subfamily of Ca2+ -permeable cation channels [21]. The seven members of this family, TRPC1-7, are sixtransmembrane domain proteins that assemble in either homoor heteromultimeric structures to get functional channels, which display several activation and modulation modalities, including different signalling pathways downstream of tyrosine kinase and G protein-coupled receptors [21,22]. TRPC channels have been associated to several calcium-dependent physiological processes in different cell types, ranging from proliferation to secretion or contractility, under both physiological and pathological conditions [23,24]; evidence for their involvement in neurite growth and guidance and in other neuronal functions regulated by calcium signals, such as survival or synaptic transmission and modulation, has emerged in the last years [24–26]. It must be pointed out that, differently from primary GnRH neurons [19], GN11 cells do not possess voltage-dependent Ca2+ channels [8], so other types of channels must be involved, and this renders this cellular model particularly interesting for the investigation of a potential role of TRPC channels in neuronal migration. Here we report that some members of the TRPC family are present in GN11 cells and their activation can account for both the basal calcium oscillations and the increased activity in the presence of FCS; their modulation by means of activators and blockers strongly influences cell motility and also, in a specific way, the proliferative rate.

Unless otherwise specified, all products were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture GN11 cells, immortalized cells obtained by genetically targeted tumorigenesis of GnRH neurons in mice [16], retaining the characteristics of immature migrating GnRH neurons [7,8], were routinely grown in monolayer at 37 ◦ C in a humidified atmosphere of 5% CO2 in air in Dulbecco’s Modified Eagle’s medium (DMEM) containing 4.5 g/l glucose and supplemented with 1 mM sodium pyruvate, 2 mM glutamine, 50 ␮g/ml gentamycin and 10% heat-inactivated fetal calf serum (FCS, Lonza, Basel, Switzerland). Subconfluent cells were routinely harvested by trypsinization and seeded on uncoated plastic dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) at a density of 3 × 103 cells/cm2 . 2.3. Quantitative real-time PCR Total RNA from GN11 cells was extracted with the RNeasy mini kit (Qiagen, Düsseldorf, Germany) and subsequently reversed transcribed to cDNA, using Ready-To-Go You-Prime first-strand beads (GE Healthcare, Buckinghamshire, UK). Triplicate cDNA samples from four independent preparation were analyzed by quantitative real-time polymerase chain reactions (qPCRs) using specific readyto-use TaqMan probe-based gene expression assays for TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (Mm00441975 m1, Mm00441984 m1, Mm00444690 m1, Mm00444284 m1, Mm00437183 m1, Mm01176083 m1, Mm00442606 m1, respectively, Applied Biosystems, Foster City, CA, USA; for more details about assay location see http://www.appliedbiosystems.com/). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ␤-actin detections (both from Applied Biosystems, Foster City, CA, USA) were used as endogenous controls and the TRPC1 signal was used as a calibrator for relative quantifications. 2.4. [Ca2+ ]i imaging

2. Materials and methods 2.1. Materials 1-[␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]1H-imidazole hydrochloride (SKF-96365) and 1-oleil 2-acetyl-sn-glycerol (OAG) were purchased from Calbiochem (San Diego, CA, USA) and dissolved in sterile water and DMSO, respectively, according to the manufacturer’s instructions. Lanthanum chloride (Sigma–Aldrich, St. Louis, MO, USA), tetrodotoxin and ␻-conotoxin (Alomone Labs, Jerusalem, Israel), were dissolved in sterile water, nifedipine (Sigma–Aldrich) in ethanol. Aspecific effects of vehicles alone at the same concentrations reached with the drugs were also tested; no significant effects on the evaluated parameters were observed. The a-TRPC1 antibody employed to block the TRPC1 subunit was a commercial rabbit polyclonal antibody obtained from Alomone Labs. This antibody was designed against a peptide (sequence QLYDKGYTSKEQKDC) corresponding to aminoacid residues 557–571 of human TRPC1 (Accession P48995), identical in mouse (NP 035773), as described [27]. This epitope, according to the models based on the hydrophobicity of the aminoacid sequence of TRPC1, should be localized near the outer mouth of the pore forming region, between the fifth and the sixth transmembrane segments [28,29]. The above sequence is however different from that of the antibody designed by Xu and Beech [29]. The TRPC1 antigen peptide used for specificity control in motility experiments was supplied by Alomone together with the a-TRPC1 antibody. The aspecific antibody was a rabbit polyclonal a-dog IgG.

Ca2+ imaging experiments were performed using the ratiometric fluorescent Ca2+ probe Fura-2-acetoxymethyl ester (Fura-2 AM; Invitrogen, Carlsbad, CA, USA). GN11 cells were plated on glass coverslips (40 mm diameter) coated with poly-l-lysine (100 ␮g/ml) at a density of 15 × 103 cells/cm2 and kept in 10% FCS DMEM for 24 h. On the day of the experiment cells were loaded with Fura-2 AM (2.5 ␮M, 30 min, 37 ◦ C), transferred to a live-cell chamber system (Bioptechs, USA) at 37 ◦ C and imaged at 1 s intervals using a monochromator system attached to an inverted microscope (Nikon TE 2000, Japan) with a 20× objective (Fluor). Images were acquired using an enhanced charge-coupled device camera (PCO, Germany) and the Metafluor software (Universal Imaging Co., Sunnyvale, CA, USA). During the experiments cells were maintained in Tyrode standard solution (154 mM NaCl, 4 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 5 mM HEPES, 5.5 mM glucose, pH adjusted to 7.35 with NaOH) circulated with a microperfusion system (inner pipette diameter 200 ␮m). For experiments in Ca2+ -free conditions, the external solution was modified, omitting the CaCl2 salt from the formulation and adding the Ca2+ chelator EGTA (0.5 mM). Fluorescence was determined from regions of interest (ROI) covering single cells. The results are expressed as R, the change of the fluorescence ratio induced by the different stimulation protocols. 2.5. Motility assays GN11 cell motility was analyzed by means of an invasion (or gap closing) assay using 2-well silicone culture-inserts (Ibidi GmbH,

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Munich, Germany), rested on the surface of uncoated plastic dishes (Falcon, Becton Dickinson). Cells were seeded at high density (60 × 103 cells/cm2 ) in 10% FCS DMEM into the wells, let adhere to the surface of the dish and form a monolayer of confluent cells. After 24 h the inserts were removed, thus creating defined cellfree gaps of 500 ␮m. Cells were washed with PBS and incubated in serum-free medium at 37 ◦ C for 1 h, following the procedure of Lentini et al. [30]. The medium was then replaced with DMEM supplemented with 5% FCS, a concentration of serum previously shown to be an efficient stimulus to induce chemokinesis in GN11 cells [7], and cells were allowed to migrate from the edges of the monolayers towards the gaps at 37 ◦ C for up to 8 h. Experiments were performed in the absence of FCS, with or without added OAG (10 ␮M), or in 5% FCS either alone or supplemented with SKF-96365 (1 ␮M), the a-TRPC1 antibody (9 ␮g/ml), the a-TRPC1 antibody preincubated with the corresponding antigen peptide (5:1 peptide:antibody), or the aspecific IgG (9 ␮g/ml). The nontoxicity of these agents at the concentrations used was excluded by viability tests. Phase contrast images of the gap regions were acquired at time 0 and after 4 and 8 h by means of a CCD video camera (CFW-1612, Scion Corp, MD, USA) connected to an inverted microscope (Eclipse TE 200, Nikon, Japan) with a 4× objective. For each insert, the distance between the two sides of the gap at different time points was measured and expressed as percentage of the original gap width (corresponding to 100%). Experiments were repeated three times and in each experiment three culture-inserts were assigned to each condition.

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condition) or supplemented with SKF-96365 at increasing concentrations (0.1–0.5–1 ␮M), the a-TRPC1 antibody (9 ␮g/ml) or the aspecific antibody (9 ␮g/ml). Four to eight wells were assigned to each condition. 24 h and 48 h later, the cell number was estimated by staining with crystal violet following a previously described colorimetric assay [31], slightly modified. Briefly, cells were fixed in 2.5% glutaraldehyde in PBS for 20 min, then stained with crystal violet (0.1% in 20% methanol), solubilized in acetic acid (10% (v/v)) and read at 595 nm in a Microplate Reader (model 550, BioRad Laboratories, Hercules, CA, USA). For each experiment, a calibration curve was set up with known numbers of cells and a linear correlation between absorbance and cell number was established. Three to five experiments were performed for each protocol; data were then pooled and expressed as mean ± SEM as percentage values of the cell number in control condition at 24 h. In order to confirm the results obtained with the crystal violet colorimetric method, another approach was also employed to study GN11 cell proliferation. Cells were cultured in 24-well plates (Falcon, Becton Dickinson) using the same experimental protocol described above, harvested by trypsinization after either 24 h or 48 h and counted with a Burker chamber. Data from four experiments were pooled and expressed as mean ± SEM. 2.7. Statistical analysis Data are expressed as mean ± SEM. Unpaired Student’s t-test or one-way ANOVA and Bonferroni post hoc test were used to evaluate statistically significant differences among groups.

2.6. Cell growth assays 3. Results Cell growth assays were employed to study the involvement of TRPC channels in GN11 cell proliferation. Optimal culture conditions to observe proliferation were assessed through a set of preliminary experiments, trying different plating densities (5 × 103 –104 cells/cm2 ) and different concentrations of FCS (5–10%) in the medium. At the end of these trials, the following protocol was established: GN11 cells were plated on uncoated plastic in 96-well plates (Falcon, Becton Dickinson) in DMEM containing 5% FCS at a density of 5 × 103 cells/cm2 . After 18 h, the medium was replaced with DMEM plus 5% FCS either alone (control

3.1. Basal and FCS-induced calcium signals When bathed in standard Tyrode solution, 27.8 ± 3.7% of GN11 cells showed spontaneous oscillations of [Ca2+ ]i (n = 513), that were completely and reversibly abolished in 100% of the cells by shifting to a calcium free extracellular solution (Fig. 1A; n = 222), pointing to a mechanism dependent on calcium influx from the extracellular medium. This oscillatory activity was unaffected by the presence of 1 ␮M tetrodotoxin, 10 ␮M nifedipine and 2 ␮M ␻-conotoxin, block-

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Fig. 1. GN11 cells show spontaneous oscillations in [Ca2+ ]i that are dependent on Ca2+ influx through non-voltage dependent calcium channels and can be enhanced by serum. A. Perfusion with a solution containing 0 Ca2+ and 0.5 mM EGTA completely suppressed spontaneous calcium transients. B. Calcium transients were unaffected by addition to the extracellular medium of 1 ␮M tetrodotoxin (TTX), 10 ␮M nifedipine (N) and 2 ␮M ␻-conotoxin (␻), blockers, respectively of Na+ and L- and N-type Ca2+ voltage-dependent channels. C. Perfusion with 5% FCS elicited calcium transients in previously silent cells (upper trace), and enhanced activity in already oscillating cells (lower trace). D. Comparison of the number of active cells (upper graph) and of the firing frequency (lower graph) between cells maintained in 0% and 5% FCS (* p < 0.05 and ** p < 0.01 vs. 0% FCS).

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ers, respectively of Na+ and L- and N-type Ca2+ voltage-dependent channels (Fig. 1B; n = 157), in accordance with previous reports that showed that these cells possess only voltage-dependent K+ channels [8]. When FCS was added to the extracellular medium, some silent cells started to generate oscillations in [Ca2+ ]i (Fig. 1C, upper trace), while in already active cells an increase in oscillatory behaviour could be observed (Fig. 1C, lower trace). The percentage of cells showing oscillations in the presence of FCS was 47.0 ± 6.8% (n = 527), a significantly greater percentage as compared to controls without FCS (see Fig. 1D, upper graph). Moreover, the frequency of oscillations showed a significant change from 0.28 ± 0.02 peaks/min in Tyrode solution to 0.37 ± 0.02 peaks/min in the presence of FCS (Fig. 1D, lower graph). 3.2. Modulation of calcium signals in GN11 cells by TRPC antagonists and agonists As a preliminary step in the evaluation of a potential contribution of TRPC channels to Ca2+ signals in GN11 cells, we performed real-time quantitative PCR experiments that showed expression of TRPC1, 2 and 5 transcripts in GN11 cells (Fig. 2A). Next, we examined the effect of the imidazole compound SKF96365, a nonselective blocker of TRPC channels already used on GnRH neurons [32–35], on calcium oscillations in GN11 cells when added to the external medium. SKF-96365 was employed at a concentration of 5 ␮M that was reported to not have any aspecific effects [36–38]. When administered to cells bathed in standard Tyrode solution, SKF-96365 abolished activity in 66% of 161 cells (Fig. 2B, upper trace); in the presence of FCS, the percentage of cells in which block of activity was detected was 64% (n = 101; Fig. 2B, lower trace). Another known blocker of TRPC channels is lanthanum (La3+ ), when used at relatively high concentrations [39,40]. When added to the extracellular medium in the presence of FCS at the concentration of 1 mM, La3+ completely abolished spiking activity in 100% of 230 cells (Fig. 2C). In an attempt to unravel specific contributions of individual members of the TRPC family to the calcium-dependent processes described above, we employed a commercial antibody raised against the extracellular side of the TRPC1 pore region (see Section 2). This antibody has previously been shown to effectively and specifically interfere with agonist-activated calcium influx in several cellular models (see e.g., [41–43]). When added to the extracellular medium, it completely blocked basal Ca2+ oscillations in 59 ± 12% of 62 cells (Fig. 2D, upper trace) and in 33 ± 2% of 74 cells showing FCS-induced signals (Fig. 2D, lower trace). An aspecific antibody failed to induce block of Ca2+ activity in basal conditions (Fig. 2D, upper trace) and in cells responding to FCS (not shown). Finally, we tested the effects of 1-oleil 2-acetyl-sn-glycerol (OAG), a diacylglycerol analogue that has been reported to be an activator of the TRPC3/6/7 subfamily [44,45], but also of TRPC1 [46,47] and TRPC2 [48,49] channels. In the presence of 100 ␮M OAG the number of cells showing activity in the absence of FCS increased by 63% (n = 43; Fig. 2E, upper trace); when added to medium containing FCS, it increased the number of cells showing activity by 33% (n = 349; Fig. 2E, lower trace). 3.3. Involvement of TRPC channels in GN11 cell motility Cell motility was analyzed by means of an invasion assay based on the use of culture-inserts. Fig. 3A shows two examples of GN11 cell migration across the gaps created by the inserts, respectively in 0% and 5% FCS. In the absence of serum after 8 h from the beginning of the experiments a very limited number of cells moved

Fig. 2. TRPC transcripts are expressed in GN11 cells and their modulation influences calcium oscillations. A. Quantitative real-time PCR showing the mRNA expression of TRPC channels (relative to TRPC1) in GN11 cells (4 independent experiments). B. 5 ␮M SKF-96365 completely and reversibly suppressed calcium oscillations recorded in basal conditions (upper trace) and in the presence of 5% FCS (lower trace). C. A comparable effect (total block of calcium signals in the presence of FCS) was observed with 1 mM La3+ . D. A block of function anti-TRPC1 antibody (9 ␮g/ml) reversibly abolished calcium oscillations in a basally active cell, while the same concentration of an aspecific IgG had no effect (upper trace). A similar effect in a cell showing FCS-induced activity (lower trace). E. 100 ␮M OAG elicited calcium oscillations in a previously silent cell kept in Tyrode solution (upper trace) and enhanced activity in a cell showing calcium oscillations in the presence of 5% FCS (lower trace).

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into the gap whereas in the presence of FCS already at 4 h the gap width is markedly reduced and nearly closed 4 h later. Moreover, we observed that in Ca2+ -free conditions (with 1.5 mM EGTA added to the medium) the migration was inhibited (data not shown). Fig. 3B shows the results of protocols with the blockers and activators used to interfere with calcium signalling in terms of occupation of the gap width. When cells were cultured in 5% FCS DMEM (upper graph), treatment with the TRPC blocker SKF-96365 (1 ␮M) significantly reduced cell motility vs. control condition at both 4 h (gap remaining 83 ± 3% vs. 93 ± 2% in control) and 8 h (60 ± 3% vs. 82 ± 4% in control). On the other hand, in the absence of serum (middle graph), 10 ␮M OAG induced a significant increase in cell migration across the gap (gap remaining at 4 h: 80 ± 1% in OAG-treated cells vs. 94 ± 2% in control cells; at 8 h: 64 ± 6% vs. 86 ± 4%). Comparable results were obtained with 100 ␮M OAG (not shown). Moreover, the anti-TRPC1 antibody significantly blocked FCS-induced cell motility at both time points of observation, while the nonspecific antibody did not have a significant effect (lower graph, gap remaining at 4 h: in 5% FCS 88 ± 1%; in 5% FCS + aspecific IgG 90 ± 2%; in 5% FCS + a-TRPC1 97 ± 1%; at 8 h: in 5% FCS 64 ± 2%; in 5% FCS + aspecific IgG 70 ± 2%; in 5% FCS + a-TRPC1 80 ± 2%). As a further control of specificity, the invasion assay was also performed in the presence of the a-TRPC1 antibody preincubated with the corresponding antigen peptide: in this case no significant reduction in FCS-induced motility was observed (lower graph, gap remaining at 4 h: 90 ± 4%; at 8 h: 60 ± 3%).

3.4. Involvement of TRPC channels in GN11 cell proliferation

Fig. 3. Effects of TRPC antagonists and agonists on GN11 cell motility. A. Examples of two experiments performed respectively in 0% and 5% FCS. B. Comparison of the invasion of GN11 cells into the gap in 5% FCS vs. 5% FCS + 1 ␮M SKF-96365 (upper), in 0% FCS vs. 0% FCS + 10 ␮M OAG (middle) and in 5% FCS alone or supplemented with either 9 ␮g/ml aspecific IgG or 9 ␮g/ml a-TRPC1 antibody alone or preincubated with the corresponding blocking peptide (lower); (upper graph: ** p < 0.01 and *** p < 0.001 vs. 5% FCS; middle graph: * p < 0.05 and ** p < 0.01 vs. 0% FCS + OAG; lower graph: ** p < 0.01 and *** p < 0.001 vs. 5% FCS).

Having evidenced the contribution of TRPC channels to Ca2+ oscillations and serum-induced motility of GN11 cells, we next investigated whether these channels may also have a role in the regulation of serum-induced proliferation, that with migratory activity characterizes this GnRH-derived cell line, by means of two different approaches, i.e., cell growth colorimetric assays and cell counting with a Burker chamber. Results obtained with the first method are reported in Fig. 4A and B. In the presence of FCS alone, significant proliferation was observed between 24 h (100 ± 7%) and 48 h (263 ± 32%) (Fig. 4A, CTR condition). When SKF-96365 was added to the medium at the concentration of 0.1 or 0.5 ␮M, no significant difference in the cell number was observed vs. control conditions, at both 24 h (101 ± 7% and 101 ± 5%, respectively) and 48 h (272 ± 37% and 242 ± 24%). On the contrary, when cells were incubated with 1 ␮M SKF-96365 the number of cells at 48 h (179 ± 11%) was significantly reduced vs. FCS DMEM alone, while at 24 h it was not significantly affected (88 ± 5%), suggesting a role for members of the TRPC family in cell cycle progression. At higher doses (3.5 ␮M and 5 ␮M) this compound exerted aspecific effects on cell survival (data not shown). Interestingly, as reported in Fig. 4B, when the anti-TRPC1 antibody and the aspecific one were used in proliferation assays, no significant reduction in cell number could be observed at both 24 h (96 ± 8% and 98 ± 12%, respectively vs. 100 ± 7% in control condition) and 48 h (220 ± 12% and 225 ± 8%, respectively vs. 222 ± 8% in control condition), pointing to a selective role of TRPC1 channels in the control of calcium-dependent motility. Similar results were obtained with the cell counting method, as reported in Fig. 4C (at 24 h: CTR 55,000 ± 4000 cells/cm2 ; 1 ␮M SKF96365 49,000 ± 2000 cells/cm2 ; a-TRPC1 58,000 ± 5000 cells/cm2 ; aspecific IgG 59,000 ± 6000 cells/cm2 ; at 48 h: CTR 111,000 ± 6000 cells/cm2 ; 1 ␮M SKF-96365 76,000 ± 3000 cells/cm2 ; a-TRPC1 110,000 ± 4000 cells/cm2 ; aspecific IgG 108,000 ± 4000 cells/cm2 ), thus confirming the involvement of TRPC channels, but not of the TRPC1 member, in the control of GN11 proliferation.

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Fig. 4. Effects of SKF-96365 and a-TRPC1 antibody on GN11 cell proliferation. A. GN11 cells were cultured in 5% FCS DMEM alone or with increasing concentrations of SKF-96365 (0.1–0.5–1 ␮M) for 24 h and 48 h; the cell number was estimated by means of a colorimetric assay. B. Similar experiments were performed in the presence of the a-TRPC1 antibody (9 ␮g/ml) and the aspecific IgG (9 ␮g/ml). C. The effects of SKF-96365 (1 ␮M), the a-TRPC1 and the aspecific antibody on proliferation were also assayed by counting detached cells in a Burker chamber (at 48 h: * p < 0.05 and ** p < 0.01 vs. CTR; in each condition ++ p < 0.01 and +++ p < 0.001 vs. 24 h).

4. Discussion Over past years, calcium dependence of neuronal migration has been extensively studied for cerebral and cerebellar cortical neurons [17,18]. Interestingly, among the few other types for which data are available, GnRH neurons show a peculiar migratory pattern [19]. GN11 cells are a good model for studying this process, as they are considered to express many properties of early stage GnRH neurons, including a strong migratory activity in the presence of serum (see e.g., [7,8]), and the modulation of this process from extracellular signals has been extensively investigated [9,12,50]. Evidence for the dependence of migration of embryonic GnRH neurons from extracellular calcium has been provided [19]. Spontaneous Ca2+

oscillations have been recorded in GN11 cells [20], but no description of the mechanisms responsible for the Ca2+ activity of these cells has been provided up to now. It must be emphasized that the Ca2+ -dependence of neuronal migration has been widely associated with voltage-dependent Ca2+ channels and NMDA receptors: in GN11 cells, the former are absent, as reported by Pimpinelli et al. [8], and confirmed by our results, and evidence for the presence of the latter is not available. In early stage GnRH neurons, calcium influx-dependent migration has been shown to be only partially dependent on voltage-dependent N-type Ca2+ channels [19]. Therefore, GN11 cells represent an interesting model of a neuronal cell whose Ca2+ oscillations, and the migratory behaviour they control, must be dependent on classes of calcium permeable channels different from voltage- and neurotransmitter-activated ones. TRPC channels are a likely candidate since they are present in mature GnRH neurons where they modulate electrical activity [32,51]. The involvement of members of the TRP superfamily in cell migration has been recently described (see e.g., [52–56]), but no data are available about the role of TRPCs, or other TRPs, in neuronal motility, differently from the abundant evidence for their role in another key process during neuronal differentiation, the growth and orientation of neuritic and axonal processes [57,58]. Here we show that the transcripts of TRPC1, 2 and 5 are expressed in GN11 cells, and provide evidence for their involvement in the migratory behaviour of a neuronal cell model. In a previous paper [59] we reported, by western blotting and immunocytochemical experiments, the presence of TRPC1, 3, 4, 5 and 6 in GN11 cells. However, due to the concerns about the specificity of some of the antibodies used (see e.g., [60]), in this paper we used a PCR approach and decided to focus only on the channels whose presence, already described, could be confirmed by the present data: TRPC1, TRPC5 (and TRPC2). This is not crucial for the interpretation of our functional data: OAG is an activator also of TRPC3 and TRPC6; La3+ , at the concentration used, is a blocker of all TRPCs, and the same holds for SKF-96365. We have found that a subpopulation of GN11 cells shows spontaneous calcium transients, in accordance with previous findings by Zaninetti et al. [20]. At variance with their data, we report that addition of serum to the extracellular medium significantly increases the percentage of active cells, and, even more relevantly, the frequency of transients. The discrepancy may be due to the fact that our data have been obtained from a much greater number of cells, thus reducing the bias due to the high variability of responses typical of many cell lines. As for the origin of the basal oscillations, either they can reflect an intrinsic property of these cells, or can be originated by an autocrine loop. This aspect requires further investigation. Both basal and serum-enhanced activity were significantly reduced by acute application of 5 ␮M SKF-96365, an agent that has been extensively used as a blocker of TRPC channels [32–35]; on the other hand, a comparably extensive set of observations has questioned its specificity [36–38,61]. However, most of the nonspecific effects have been reported at doses at least one order of magnitude above 5 ␮M, the higher concentration used in our experiments. Moreover, some of the effects refer to voltage-dependent calcium channels [38,62] that are absent in these cells. Further support for a potential role of TRPC channels in the generation of calcium oscillations comes from the use of other modulators of these channels: 1 mM La3+ (that at this concentration is considered to act as a blocker of TRPCs, see [39,40]) completely abolished activity in 100% of cells, and an a-TRPC1 blocking function antibody blocked calcium signals in a significant subpopulation of cells, both in the absence and in the presence of FCS. On the other hand the diacylglycerol analogue OAG (100 ␮M), that in addition to the members of the TRPC3/6/7 subfamily [44,45],

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has been reported to activate also TRPC1 [46,47] and TRPC2 channels [48,49], strongly increased activity. These observations are in close agreement with the functional data obtained by interfering with motility and proliferation using the same pharmacological tools. SKF-96365 (1 ␮M) and the a-TRPC1 antibody (9 ␮g/ml) strongly and specifically reduced FCSinduced motility, while OAG (10 ␮M) induced a strong increase in the same process when applied in the absence of serum. While the effects of FCS on migration are quite dramatic, those on the percentage of cells generating calcium oscillation are not as strong: however, the increase in frequency of spikes is enhanced, and this could be the key signal in controlling motility. It is known that frequency of calcium signals can code information relevant to several cellular processes, such as gene expression [63], neuronal differentiation [64] and also neuronal migration [17]. Interestingly, the fact that neuronal motility may depend on calcium influx through non voltage-activated calcium channels may explain the observation by Pimpinelli et al. [8], that depolarization with high KCl reduced migration of GN11 cells. Given that these cells do not have voltage-dependent calcium channels, depolarization will straightforwardly reduce the Ca2+ gradient and consequently the passive calcium influx. Finally, we observed a reduction in serum-induced proliferation (that together with motility is a hallmark of this cell line) following treatment with a low concentration of SKF-96365 (1 ␮M). Understanding which of the members of the TRPC family expressed in GN11 cells are actually involved in the processes described above is made difficult by the lack of specific blockers and activators. In order to interfere with TRPC1 channels we used a block of function antibody, raised against the extracellular side of the pore region of the TRPC1 subunit [27,28]. In our hand this a-TRPC1 antibody has proven to be quite specific, since the same antibody preincubated with the corresponding blocking peptide as well as a control aspecific IgG failed to interfere with Ca2+ signals and functional parameters. The results showed that the a-TRPC1 antibody can abolish both basal and serum-induced calcium oscillations in a subpopulation of cells, and significantly reduces cell migration. Interestingly, it does not affect proliferation, thus providing further evidence for its specificity. It can be deduced that different TRPC members may be involved in the control of specific, even if coordinated, developmental events. In conclusion, our data provide evidence for the involvement of calcium-permeable channels of the TRPC family in the control of the functional properties of a cell line widely used as a model of neuroendocrine precursors. Moreover, we report for the first time a role for TRPC channels in neuronal motility. More refined approaches, such as gene silencing, will be needed to confirm these results. It must be considered that other calcium permeable channels, such as other members of the TRP superfamily may be involved in this process; however, a new link between motogens, calcium signalling and neuronal motility has been revealed.

Acknowledgements Support from the Compagnia di S. Paolo to Paolo Ariano (2008.21.91 P.A.) and to the NIS Centre of Excellence is gratefully acknowledged. Part of the work was supported by grants from the Belgian Federal Government (IUAP P6/28), the Research Foundation-Flanders (F.W.O.) (G.0565.07 and G.0686.09, G.O., B.N.) and the Research Council of the KU Leuven (GOA 2009/07 and EF/95/010, G.O., B.N.).

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