ATP stimulates chemokine production via a store-operated calcium entry pathway in C6 glioma cells

All chemicals were purchased from Sigma (St.Louise, MO) unless otherwise stated. Two inhibitors of SOC were employed in this work; gadolinium [17, 18] and SKF96365 [19].

C6 glioma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells from passage number 39-59 were used in this work. Cells were cultured in Kaighn's modification of Ham's F12 medium (F12K) with 2 mM l-glutamine modified by ATCC to contain 1.5 g/l sodium bicarbonate. The medium was then supplemented with 15% horse serum, 2.5% fetal bovine serum, 0.5 μg/ml fungizone (Invitrogen: GIBCO, Grand Island, NY) and 0.02 mg/ml gentamicin (Invitrogen: GIBCO). Cells were maintained in 100 mm culture dishes (SARSTEDT, Newton, NC) at 37°C in a humidified 5% CO₂ air atmosphere.

The detailed procedure for calcium imaging was carried out as published [20]. Briefly, cultured C6 glioma cells were incubated with fura-2 acetoxymethyl ester (fura-2AM at 1 μM; Molecular Probes, Eugene, OR) and pluronic acid (at 1 μM) in normal physiological saline solution (PSS) for 20 min at room temperature (20-22°C). Cells were then washed with PSS solution containing (in mM): 126 NaCl, 5 KCl, 1 CaCl₂, 1.2 MgCl₂, 10 HEPES and 10 glucose (pH 7.4). In some experiments, Ca²⁺-free PSS was used; this solution had the same composition as PSS with the exception that EGTA was added (at 1 mM) with no CaCl₂. Coverslips were placed in a perfusion chamber mounted on an inverted microscope (Zeiss, Jena, Germany) and fluorescence was measured through a 40× quartz objective lens. Alternating wavelengths (340/380 nm) of ultraviolet light were applied at 6-s intervals for excitation and fluorescence signals were measured at 510 nm of emission light. Signals were acquired from a digital camera (DVC-1310, DVC Co. Austin, TX) and were processed using an imaging system (Empix, Mississauga, ON, Canada) to determine ratios of the 340 and 380 nm intensities which were used as quantitative measures of fluorescence levels in this work.

C6 glioma cells (8 × 10⁵ cells/well) were plated in serum-free media and the experiment was carried out the following day with addition of ATP (100 μM) in the absence or presence of SKF96365 (25 μM) or gadolinium (1 μM) for 4 h. Total RNA was isolated using TRIzol (Invitrogen: GIBCO) and then processed for the first strand complementary DNA (cDNA) synthesis using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen: GIBCO). The cDNA products were then amplified by PCR using a GeneAmp thermal cycler (Applied Biosystems, Foster City, CA). Specific sense and antisense primers with the expected product sizes are shown in Table 1. PCR conditions were as follows: initial denaturation at 95°C for 6 min followed by a 25- to 35- cycle amplification program consisting of denaturation at 95°C for 45 s, annealing at 55-60°C for 1 min and extention at 72°C for 1 min. A final extention was carried out at 72°C for 10 min. For P2YR expression study, the PCR cycle was set at 35 cycles for all subtype receptors. Amplification of the constitutively expressed enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reaction standard. The amplified PCR products were identified using 1.5% agarose gels containing ethidium bromide (final concentration 0.5 μg/ml) and visualized under ultraviolet light. The intensities of each band were measured by densitometry using NIH Image J 1.37b software (National Institute of Health, Bethesda, MD) and expressed as relative mRNA levels (mRNA levels normalized to GAPDH).

C6 cells were plated using serum-free media in a 24-well plate at 3 × 10⁵ cells/well and were allowed to acclimate for 24 h before starting the experiment. ATP was applied to the cells with or without gadolinium for 48 h and culture media were collected and analyzed with rat MCP-1 and rat IL-8 (GRO/KC) ELISA kit (Peprotech, Rockey Hill, NJ) according to the manufacturer's protocol. The detection limits were 47 and 16 pg/ml for MCP-1 and IL-8, respectively.

Application of ATP (100 μM) to C6 glioma cells resulted in an early phase of increased [Ca²⁺]i which rapidly declined and was followed by a sustained component of response (Fig. 1A). In the presence of Ca²⁺-free PSS (0 PSS), ATP administration led to a single phase of [Ca²⁺]i mobilization representing only the rapid portion of the response (Fig. 1B). These results suggested ATP binding to a P2YR activating an inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular release of Ca²⁺ and a subsequent entry of divalent ion through SOC [21, 22]. To test this possibility two inhibitors of SOC, gadolinium and SKF96365, were applied prior to ATP stimulation. The concentrations of gadolinium (at 1 μM) [18] and SKF96365 (at 25 μM) [23, 24] were chosen according to ones used in previous studies which were found effective for SOC inhibition. Representative ATP responses are presented with pre-treatment (3 min) with gadolinium (Fig. 1C) or SKF96365 (Fig. 1D). Exposure of C6 to either SOC inhibitor yielded a pattern of ATP response close to that observed in Ca²⁺-free PSS (Fig. 1B) with attenuation of the prolonged component of [Ca²⁺]i. Peak amplitudes of [Ca²⁺]i responses did not appreciably differ between control or the different treatments. Overall results are summarized in Fig. 1E (N = 4/treatment) and show durations (measured at half-maximal of peak value) of ATP responses were similar between Ca²⁺-free PSS and treatments with the two SOC inhibitors. Respective inhibitions of response durations relative to control ATP response were 44% (0 PSS), 49% (gadolinium) and 55% (SKF96365).

RT-PCR experiments were undertaken to determine the expression of P2YR in unstimulated and ATP-stimulated C6 cells. The study examined only those P2YR known to be G protein coupled to IP₃-dependent intracellular stores [21, 22] and included P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptors. Typical expression of these subtype receptors are presented in Fig. 2 for control and ATP stimulation of C6 glioma for 4 h. Expression of mRNA for any of the subtype P2YR were unchanged between control and cells exposed to ATP treatment. The levels of band intensities occurred in the following order: P2Y₂R > P2Y₁R > P2Y₆R > P2Y₄R. Consistent results were obtained in three additional experiments with the intensity of P2Y₂R exceeding levels for the other subtype P2YR for both control and ATP-stimulated glioma. Exposure of cells to longer-term ATP solution (48 h) had no significant effect to alter expression of any of the subtype P2YR (data not shown).

Chemokines, such as MCP-1 and IL-8, released from ATP-stimulated glioma could act to mobilize immune cells nearby tumor microenvironments. Representative RT-PCR for these two chemokines is presented in Fig. 3A for the different treatments (4 h exposure) applied to C6 glioma cells. Application of ATP (100 μM) markedly enhanced expression of MCP-1 and IL-8 relative to control. Treatments of SKF96365 (25 μM) or gadolinium (1 μM) with ATP attenuated chemokine expression compared with ATP alone. Application of SKF96365 or gadolinium alone showed mRNA for MCP-1 and IL-8 similar to control. Quantification for the relative expression of the two chemokines with the different treatments (N = 4/treatment) of C6 glioma is presented in Fig. 3B. ATP stimulation increased expression of MCP-1 by 4.4-fold and IL-8 by 3.2-fold relative to control. Expression of MCP-1 was significantly reduced with SKF96365 (by 78%) and gadolinium (by 64%) treatment of ATP-stimulated C6 relative to ATP application alone. The corresponding decreases for IL-8 were 46% with SKF96365 and 40% with gadolinium with both values representing significant effects of the SOC antagonists.

We next examined if the enhancing effect of ATP and the inhibitory effect of SOC inhibitors on chemokine expression were translated at the protein levels. Preliminary ELISA studies showed that production of both MCP-1 and IL-8 was detectable as early as 4 h but in the low range of detection limits for up to 24 h after ATP stimulation. Subsequent experiments used a 48 h exposure of C6 glioma to ATP with gadolinium studied as an SOC inhibitor. Sustained exposure of C6 cells to SKF96365 (25 μM) caused cytotoxicity to cells thus precluding use of this compound for longer-term treatment. The overall results (N = 4/treatment) show MCP-1 production was increased by 131% with ATP stimulation compared with control (Fig. 4A). Inclusion of gadolinium with ATP significantly attenuated MCP-1 (by 64%) relative to ATP alone. Levels of IL-8 were also enhanced by ATP (by 92%) compared to control (Fig. 4B). Addition of gadolinium with ATP was effective in reducing levels of IL-8 by 54% compared to ATP applied alone.