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Published as doi: 10.1096/fj.06-6016fje.
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(The FASEB Journal. 2006;20:2381-2383.)
© 2006 FASEB

Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells

Wei-Chiao Chang, Charmaine Nelson and Anant B. Parekh1

Department of Physiology, University of Oxford, Oxford, England, UK

1Correspondence: Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK E-mail: anant.parekh{at}physiol.ox.ac.uk

ABSTRACT

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-dependent enzyme that mediates agonist-dependent arachidonic acid release in most cell types. Arachidonic acid can then be metabolized by the 5-lipoxygenase enzyme to generate the proinflammatory signal leukotriene C4 (LTC4). Here we report that Ca2+ entry through store-operated CRAC (Ca2+ release-activated Ca2+) channels activates the extracellular signal-regulated kinases (ERKs), members of the mitogen-activated protein kinase family, within minutes and this is necessary for stimulation of cPLA2. Ca2+ entry activates ERK indirectly, via recruitment of Ca2+-dependent protein kinase C {alpha} and ßI. Ca2+ influx also promotes translocation of cytosolic 5-lipoxygenase to the nuclear membrane, a key step in the activation of this enzyme. Translocation is dependent on ERK activation. A role for gene activation is shown by the finding that CRAC channel opening results in increased transcription and translation of c-fos. Inhibition of ERK activation failed to prevent c-fos expression. Our results show that CRAC channel activation elicits short-term effects through the co-coordinated regulation of two metabolic pathways (cPLA2 and 5-lipoxygenase), which results in the generation of both intra- and intercellular messengers within minutes, as well as longer term changes involving gene activation. These short-term effects are mediated via ERK, whereas, paradoxically, c-fos expression is not. Ca2+ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses.—Chang, W.-C., Nelson, C., Parekh, A. B. Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells.


Key Words: store-operated influx • 5-lipoxygenase • protein kinase

THE 85 KD CALCIUM-DEPENDENT cytosolic phospholipase A2 (cPLA2) is expressed in most cells, where it hydrolyzes membrane phospholipids containing arachidonate at the sn-2 position to release arachidonic acid on cell stimulation (1) . Arachidonic acid is a pleiotropic intracellular messenger regulating ion fluxes across the plasma membrane (2 , 3) , protein kinases (4) , and apoptosis (5) . It is also the precursor of potent proinflammatory eicosanoids like the cysteinyl leukotrienes (6) . Modest increases in cytoplasmic Ca2+ concentration promote translocation of cPLA2 to intracellular membranous systems, notably the Golgi, endoplasmic reticulum, and perinuclear membrane, where it can access its phospholipid substrates (7 8 9) . Binding to membranes is mediated by the Ca2+-dependent phospholipid binding (or C2) domain located in the N-terminal of the enzyme (10 , 11) . Recent evidence suggests that ceramide-1-phosphate binds to the C2 domain in a Ca2+-dependent manner and may be involved in the translocation process (12) .

Although a rise in cytoplasmic Ca2+ is necessary for cPLA2 translocation and binding to membranes, the catalytic activity of cPLA2 is not dependent on Ca2+, at least following physiologically relevant Ca2+ rises (1 , 10 , 13) . Instead, phosphorylation of the enzyme is important (1) but the underlying mechanism appears to be complex. Extracellular signal-regulated kinases (ERKs), which are members of the mitogen-activated protein kinase family, exist as two isoforms called ERK1 and ERK2. ERK1/2 phosphorylate cPLA2 on Ser-505 and this is necessary for cPLA2-mediated arachidonic acid release following stimulation with a variety of agonists in a diverse array of cell types (14 15 16) . Detailed analysis of phosphorylation of cPLA2 in the baculovirus/insect expression system revealed that the enzyme is in fact phosphorylated on four serines [437, 454, 505, and 727; (17) ]. In human platelets and HeLa cells, cPLA2 is only phosphorylated on Ser-505 and Ser-727, following stimulation with agonists (18) . Ser-727 is phosphorylated by MNK1-related protein kinases, which are activated by p38 mitogen-activated protein kinases (19) , and expression of phosphorylation site mutants in which Ser-505 and Ser-727 were replaced by alanines indicated that phosphorylation of both sites was important for the Ca2+ ionophore A23187 (1 µM) to stimulate arachidonic acid release. A high concentration of ionophore (10 µM), which increased the Ca2+-dependent fluo-3 signal 8- to 11-fold, indicating a large rise in cytoplasmic Ca2+, evoked arachidonic acid release even in cells expressing the mutant cPLA2 constructs (19) . Indeed, in some cell types [thrombin-stimulated platelets (20) and rat-liver macrophages (21) ], it has been reported cPLA2 can be activated with no requirement for Ser-505 phosphorylation. In rat macrophages, Ser-505 phosphorylation-independent activation of cPLA2 was observed following stimulation with micromolar concentrations of the Ca2+ ionophore A23187, applied for several minutes. However, this stimulation protocol raises cytoplasmic Ca2+ to grossly nonphysiological levels. It is therefore conceivable that both ERK and MNK1-mediated phosphorylation, as well as elevation of Ca2+ in the physiological range are needed to stimulate cPLA2 effectively, but extremely large cytoplasmic Ca2+ signals bypass the requirement for the ERK pathway.

Phosphorylation of cPLA2 by ERK greatly increases the ability of Ca2+ mobilizing agonists to liberate arachidonic acid (22) . Agonists that transiently increase intracellular Ca2+ but do not activate ERK fail to stimulate cPLA2 in macrophages. Similarly, agonists that activate ERK but fail to raise intracellular Ca2+ are also ineffective. However, the agonists act synergistically in that combining them results in cPLA2 activation and arachidonic acid release (22) . Moreover, promoting phosphorylation of cPLA2 by inhibiting serine/threonine phosphatases with okadaic acid (OA) is sufficient to activate arachidonic acid release even at resting cytoplasmic Ca2+ levels, although the effect is blunted by lowering basal Ca2+ (22) . Hence cytoplasmic Ca2+ rises in the physiological range and ERK activation are both needed for effective stimulation of cPLA2 in many cells. One intriguing model for how these two stimuli might interact is that the Ca2+ rise enables the amino-terminal C2 domain to insert into the membrane, but phosphorylation of Ser-505 is required for aliphatic amino acids at the cPLA2 active site rim to penetrate the membrane, thus positioning the active site for optimal catalysis (23) .

One important question concerns a potential link between cytoplasmic Ca2+ and ERK activation in stimulating cPLA2. ERK is activated following dual phosphorylation of threonine and tyrosine residues by the upstream kinases MEK1/2. However, neither ERK nor MEK1/2 have any intrinsic Ca2+-dependence. Ca2+ and ERK are therefore considered to function independently, with Ca2+ promoting binding of cPLA2 to intracellular membranes while ERK phosphorylation results in activation of the catalytic site. Alternatively, an intriguing possibility is that there might be a Ca2+-dependence to ERK activation. If so, then cytoplasmic Ca2+ would regulate cPLA2 in two ways: translocation of the enzyme to intracellular membranes as well as its subsequent activation via recruitment of the MEK/ERK signaling cascade. In some cell types, a rise in cytoplasmic Ca2+ concentration can activate ERK, although the underlying mechanism remains unclear (24 25 26 27) .

Once released from phospholipids, arachidonic acid can be metabolized by the 5-lipoxygenase enzyme to generate potent proinflammatory signals, like leukotriene C4 [LTC4; (6) ]. We have recently shown that Ca2+ entry through store-operated CRAC channels activates cPLA2 and that the liberated arachidonic acid is metabolized by the 5-lipoxygenase enzyme to generate LTC4 (28) . Like cPLA2, 5-lipoxygenase has a C2 domain in its N-terminal, which is necessary for translocation of the enzyme to the nuclear membrane (29 30 31) . Here, it binds to the 5-lipoxygenase activating protein FLAP, resulting in enzyme activation (6) . However, despite its importance, little is known about the pathways regulating 5-lipoxygenase translocation.

In this study, we demonstrate that Ca2+ entering through CRAC channels activates the MEK/ERK pathway through stimulation of Ca2+-dependent protein kinase C. We also find that ERK is needed for translocation of the 5-lipoxygenase to the nuclear membrane. Hence, ERK is a versatile transducer of CRAC channel activation and translates this into coordinated regulation of two metabolic pathways (cPLA2 and 5-lipoxygenase), resulting in the generation of both intra- and intercellular messengers within minutes. Because ERK activation can initiate gene transcription, we have also examined whether CRAC channel opening stimulates gene expression via ERK. We find that transient Ca2+ entry through CRAC channels increases both transcription and translation of the immediate early gene c-fos. Intriguingly, this latter pathway does not seem to be mediated by ERK. Ca2+ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses.

MATERIALS AND METHODS

Cell culture
RBL-1 cells were bought from American Type Culture Collection (ATCC). Cells were cultured (37°C, 5% CO2) in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS, 2 mM L-glutamine, and penicillin-streptomycin, as described previously (28 , 32) . For Ca2+ imaging and patch-clamp experiments, cells were passaged (using trypsin) onto glass coverslips and used 24–48 h after plating.

Ca2+ imaging
Ca2+ imaging experiments were performed using the IMAGO CCD camera-based system from TILL Photonics, as described previously (28 , 33) . Cells were alternately excited at 356 and 380 nm (20 ms exposures) using a Polychrome Monochromator, and images were acquired every 2–3 s. Images were analyzed offline using IGOR Pro. Cells were loaded with Fura-2-AM (2 µM) for 40 min at room temperature in the dark and then washed three times in standard external solution of composition (in mM) NaCl 145; KCl 2.8; CaCl2 2; MgCl2 2; D-glucose 10; HEPES 10, pH 7.4; with NaOH. Cells were left for 15 min in the dark to allow further deesterification. Ca2+ signals are presented as the change in resting ratio (356/380) prior to stimulation, {Delta}R.

Patch clamp recordings
Whole cell patch-clamp experiments were performed as described (32 , 33) . Sylgard-coated, fire-polished patch pipettes filled with a solution that contained 145 mM cesium glutamate; 8 mM NaCl; 1 mM MgCl2; 2 mM Mg-ATP; 10 mM HEPES; 10 mM EGTA; 30 µM InsP3 (0.03); and 2 µM thapsigargin, pH 7.2, with CsOH. Pipette resistance was ~5 MOhms when placed in an external solution containing 145 mM NaCl; 2.8 mM KCl; 10 mM CaCl2; 2 mM MgCl2; 10 mM CsCl; 10 mM D-glucose; 10 mM HEPES, pH 7.4, with NaOH. A correction of +10 mV was applied for the subsequent liquid junction potential that arose from the glutamate-based pipette solution. ICRAC was measured at –80 mV from voltage ramps (–100 to +100 mV lasting 50 msec) applied at 0.5 Hz, from a holding potential of 0 mV and normalized to cell size (capacitance). Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitized at 100 µs. Capacitative currents were compensated before each ramp. Leak currents were subtracted by averaging two ramp currents obtained after break-in from all subsequent recordings.

Immunofluorescence
Cells were fixed in 4% paraformaldehyde in phosphate buffer for 30 min at room temperature, after stimulation with thapsigargin. All the washes used 0.01% PBS (PBS; in mM: NaCl 137, KCl 2.7, Na2HPO4 8, KH2PO4 1). The cells were blocked with 2% BSA and 10% goat serum for 1 h. 5-Lipoxygenase was visualized using a monoclonal mouse IgG1 antibody (Ab) (BD Transduction Laboratories, Oxford, U.K.; used at a dilution of 1:3000). Protein kinase C {alpha} and ßI were detected using antibodies from Santa Cruz Biotechnology (Santa Cruz, CA; both used at 1:2000 dilution). Anti-5-lipoxgenase, antiprotein kinase C {alpha} and antiprotein kinase C ßI were used in carrier (0.2% BSA, 1% goat serum) and left overnight at 4°C. The secondary anti-mouse IgG was a HandL chain specific (goat) fluorescein conjugate (excitation at 495 nm, emission at 515 nm). This was used at 1:2000 in PBS for 2 h at room temperature. The cells were mounted in Vectashield mounting medium containing a propidium iodide counterstain for DNA, (excitation 535 nm, emission 615 nm). Images were obtained using a Leica confocal microscope.

Preparation of cell lysates
Attached cells from 10 cm plastic dishes were washed twice with PBS and lysed with PBS buffer containing 0.5% Triton X-100 (Sigma, St. Louis, MO, USA) and protease cocktail inhibitor (Sigma), as described (28) . Lysates were centrifuged at 8000 rpm for 5 min, and the supernatants were collected and stored at –70°C until used.

RT-polymerase chain reaction (RT-PCR)
Total RNA was extracted from RBL cells by a RNeasy Mini Kit (Qiagen, West Sussex, UK). A reverse-transcriptase reaction was performed on 3 µg of extracted total RNA using the Superscript II Kit (Invitrogen, Paisley, UK), according to the manufacturer’s instructions. Following cDNA synthesis, PCR was then performed with primers specific for rat c-fos (sense, 5'-AGCCGACTCCTTCTCCAGCAT-3', and antisense, 5'-CAGATAGCTGCTCTACTTTGC-3', with expected product size of 298 bp) and Beta Actin (sense, 5'-TTGTAACCAACTGGGACGATATG-3', and antisense, 5'-GATCTTGATCTTCATGGTGCTAGG-3', with expected product size of 764 bp). Thirty-two cycles of PCR were performed, with each cycle consisting of denaturation at 94°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 45 s. The PCR products were electrophoresed through an agarose gel and visualized by ethidium bromide staining.

Western blotting
Total cell lysates (40 µg) and nuclear extract (60 µg) were analyzed by SDS-PAGE on a 10% gel. Membranes were blocked with 5% nonfat dry milk in PBS plus 0.1% Tween 20 (PBST) buffer for 2 h at room temperature. Membranes were washed with PBST three times and then incubated with primary Ab for 1 h at room temperature. Antiphospho-ERK Ab, which recognizes dual phosphorylated (i.e., active) ERK, was from New England BioLab (Hartfordshire, UK) and used at 1:2000 dilution. Total ERK 2, c-fos, and antiphospho (serine 505)-cPLA2 antibodies were from Santa Cruz Biotechnology, used at dilutions of 1:5000 (ERK2) and 1:1000, respectively, and secondary Ab was rabbit IgG (1:2500). The membranes were then washed with PBST again and incubated with 1:2000 dilution of peroxidase-linked anti-mouse IgG from Amersham Bioscience (Buckinghamshire, UK) for 1 h at room temperature. After washing with PBST, the bands were detected by an enhanced chemiluminescence (ECL)-plus Western blotting detection system (Amersham Biosciences).

Nuclear extract
RBL cells were washed twice with PBS and scraped in 0.5 ml of PBS. Cells were collected by centrifuging at 12,000 rpm for 10 s and resuspended in 200 µl of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, and 0.5 mM EDTA] at 4°C for 10 min. Nuclei were pelleted by centrifugation at 12,000 rpm for 10 s and resuspended in 100 µl of buffer B [20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol (v/v)] at 4°C for 20 min. Supernatants were collected following centrifugation at 12,000 rpm for 2 min and stored at –80°C until use. Buffer A and buffer B described above contained 0.5 mM phenylmethylsulphonyfluoride (PMSF), 1 mM orthovanadate, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin.

Quantification of gels
The films were scanned on a flatbed scanner, and the relative band intensities were digitized and analyzed by using UN-SCAN-IT software (Silk Scientific, Utah, USA).

[3H]-Arachidonic acid release
Cells were prelabeled with 0.25 µCi/ml [3H]-Arachidonic acid in DMEM for 1.5 h at 37°C, as described previously (28) . Cells were then washed twice with serum-free DMEM to remove unincorporated [3H]-arachidonic acid. Thapsigargin with 0.5% fatty acid–free BSA was added for different times (see text). Medium was collected and centrifuged at 25g for 5 min to remove any floating cells. Radioactivity in the supernatant was measured. The amount of [3H]-arachidonic acid released into the medium was expressed as a percentage of the total [3H]-arachidonic acid uptake.

LTC4 measurements
Following stimulation of attached cells with thapsigargin, the supernatant was collected and LTC4 levels were measured by enzyme immunoassay (EIA) (Cayman Chemicals, Ann Arbor, MI, USA) as described previously (28) . In brief, 50 µl of supernatant, leukotriene C4 acetylcholinetransferase, and leukotriene C4 antiserum were added to each EIA well. Following incubation for 18 h at room temperature, the wells were emptied and rinsed five times with EIA washing buffer. Ellman’s reagent (200 µl; prepared fresh) was added to each well, and the plate was placed on an orbital shaker for 1.5 h in the dark. Plate absorbance was measured at a wavelength of 405 nm.

Statistical analysis
Data are presented as the mean ± SEM. Statistical significance was considered as P < 0.01, using Student’s t test, and is denoted by *.

RESULTS

Ca2+ entry through CRAC channels stimulates ERK and this is necessary for arachidonic acid release and LTC4 secretion
cPLA2 can be activated following phosphorylation of Ser-505 by the p42/p44 mitogen activated protein (MAP) kinases ERK1/2 (8 , 14 , 15 , 18) . ERK1/2 is activated following dual phosphorylation of critical threonine and tyrosine residues by the upstream MAP kinase kinases MEK1/2 (34 , 35) . We used a monoclonal antibody (mAb) that specifically recognizes dually phosphorylated (i.e., active) ERK, an approach that has been used widely to monitor ERK activation (24 , 36) . Stimulation with thapsigargin for 4 min in the presence of external Ca2+ resulted in an increase in the levels of active ERK (Fig. 1 Aa, upper panel). Activation was much less pronounced following stimulation in the absence of external Ca2+ (Fig. 1Aa ) or when thapsigargin was applied in external Ca2+ but in the presence of the CRAC channel blocker 2-APB [30 µM; (37) ; Fig. 1Ab ]. Furthermore, mitochondrial depolarization (evoked either by the protonophore FCCP or the respiratory chain inhibitor rotenone), which impairs CRAC channel activity and the subsequent cytoplasmic Ca2+ rise (33 , 38) , reduced ERK activation following stimulation with thapsigargin (Fig. 1Ac ). Inhibition of the mitochondrial ATP synthase with oligomycin was much less effective (Fig. 1Ac ). Hence Ca2+ entering through CRAC channels can activate the MEK/ERK pathway and this is regulated by the energized state of mitochondria. U0126, which impairs ERK activation by blocking MEK1 and MEK2 (39) , suppressed ERK activation by thapsigargin (Fig. 1Aa ). These results are in agreement with a report from jurkat T cells demonstrating that stimulation with thapsigargin activated ERK, although the Ca2+ influx pathway in that study was not identified (40) .


Figure 1
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  		Figure 1. Ca2+ entry through CRAC channels activates cPLA2 and LTC4 secretion through the ERK pathway. Aa) Western blots show that stimulation with 2–5 µM thapsigargin (4 min) results in the phosphorylation of ERK1/2 (denoted in upper panel as ERK1/2-P), indicative of ERK activation. Phosphorylation is prevented by removing external Ca2+. The last lane shows that the MEK1/2 inhibitor U0126 (20 µM pretreatment for 15 min) suppresses thapsigargin-induced ERK phosphorylation. Total ERK2 levels are shown (lower panel), as an indicator of constant loading of protein into each lane. Ab) Western blot showing thapsigargin-evoked ERK activation is prevented by the CRAC channel blocker 2-APB (30 µM). Loading control is shown in lower panel. Ac) Mitochondrial depolarization with either FCCP (5 µM) or rotenone (1 µg/ml) reduces ERK phosphorylation (both applied in the presence of 1 µg/ml oligomycin). Oligomycin alone had only a weak effect. Loading control is shown in the lower panel. B) Thapsigargin stimulates phosphorylation of cPLA2 on Ser-505. Upper panel shows a Western blot with a mAb that specifically recognizes Ser-505 cPLA2. The lower panel plots quantitative analysis from three such gels. Thapsigargin induced a significant increase in Ser-505 phosphorylation compared with control (P<0.01) and the increase was substantially reduced by either U0126 or PD98059 (P<0.01 for both cases). C) U0126 and PD98059 inhibit arachidonic acid release following CRAC channel opening. D) U0126 inhibits LTC4 secretion in a concentration-dependent manner. E) Cytoplasmic Ca2+ signals arising from store-operated Ca2+ entry are unaffected by U0126 (20 µM; 15 min pretreatment; 33 cells for control and 37 cells for U0126). Cells were pretreated with thapsigargin in Ca2+-free solution for 10 min before external Ca2+ was readmitted. F) The amplitude of ICRAC was not affected by U0126 (5 cells for each condition). *Denotes P < 0.01.

ERK phosphorylates cPLA2 on Ser-505, and this phosphorylation is required for enzyme activation. The upper panel of Fig. 1B shows that stimulation with thapsigargin resulted in significant phosphorylation of cPLA2 on Ser-505 (detected with a mAb recognizing only this epitope), and this was suppressed by both U0126 and the structurally distinct MEK blocker PD98059 (41) . Quantitative analysis from three such gels is shown in the lower panel of Fig. 1B . As reported by others, there is some phosphorylation of Ser-505 in nonstimulated cell and this is reduced by inhibiting ERK activation (Fig. 1B ). Consistent with activation of cPLA2, thapsigargin stimulated the release of arachidonic acid and this was suppressed by both U0126 and PD98059 (Fig. 1C ). Thapsigargin-evoked arachidonic acid release was used to synthesize LTC4 (Fig. 1D ), and this was inhibited by U0126, with an IC50 in the nanomolar range (Fig. 1D ). Importantly, inhibition of MEK1/2 failed to alter the size of the cytoplasmic Ca2+ signal arising from store-operated Ca2+ entry (Fig. 1E ) or the extent of ICRAC (Fig. 1F ). Therefore, inhibition of ERK1/2 activation impairs cPLA2 activation and subsequent LTC4 secretion without affecting the cytoplasmic Ca2+ signal.

Ca2+ entry through CRAC channels stimulates translocation of Ca2+-dependent protein kinase C
Because the MEK/ERK pathway is not thought to have any intrinsic Ca2+ dependence, we suspected that Ca2+ entry through CRAC channels activated MEK/ERK1/2 via an intermediary Ca2+-sensitive molecule. MEK is often activated by Raf-1, which in turn can be activated through a protein kinase C-dependent pathway (42 43 44) . RBL cells express five protein kinase C isozymes (45) . Two of these are the conventional (Ca2+-dependent) {alpha} and ß isoforms (of which there are two variants: ßI and ßII), two are novel isozymes ({delta} and {epsilon}), and one is atypical ({zeta}). We reasoned that if a protein kinase C isozyme was involved in translating CRAC channel activity to ERK activation, it would likely involve a conventional Ca2+-dependent isoform. Ca2+-dependent protein kinase C isoforms translocate to the plasma membrane on stimulation and this migration is required for enzyme activation. In resting RBL-1 cells, protein kinase C {alpha} and ßI are distributed mainly in the cytoplasm (Fig. 2 Aa and Ba). After stimulation with thapsigargin, both isoforms translocate to the cell periphery (Fig. 2Ab and Bb ). Importantly, the translocation is suppressed either by removing external Ca2+ (Fig. 2Ac ) or inhibiting CRAC channels with 2-APB (Fig. 2Ad ). Chronic exposure to phorbol ester selectively down-regulates conventional protein kinase C isoforms in RBL cells (45) . Consistent with this, we found that expression of both protein kinase C {alpha} and ßI were dramatically reduced following exposure to 1 µM PMA for 18 h (data not shown). This resulted in substantial inhibition of both arachidonic acid release (Fig. 2C ) and LTC4 secretion (Fig. 2D ). Shorter exposure times (3–6 h) to a low concentration of PMA (25 nM), which partially down-regulates the two Ca2+-dependent protein kinase C isoforms (45) , reduced ERK activation (data not shown) and significantly decreased arachidonic acid release induced by thapsigargin (by 59±6%). Collectively, the results demonstrate a major role for the protein kinase C {alpha} and ß isozymes in linking store-operated Ca2+ entry through CRAC channels to cPLA2 activation via ERK.


Figure 2
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  		Figure 2. Protein kinase C {alpha} and ßI translocate to the cell periphery following Ca2+ entry through CRAC channels. A) Confocal images showing that protein kinase C ßI is cytosolic at rest (Aa), translocates to the periphery after stimulation with thapsigargin (in external Ca2+) for 4 min (Ab) and this is blocked by removing external Ca2+ (Ac) or applying 2-APB (Ad). B) Protein kinase C {alpha} is primarily cytoplasmic at rest (Ba) but translocates to the cell periphery following stimulation (Bb). C, D) Chronic exposure to PMA (1 µM) for 18 h substantially reduces thapsigargin-evoked arachidonate release and LTC4 secretion, respectively.

Inhibition of conventional protein kinase C suppresses ERK activation, arachidonic acid generation, and LTC4 secretion
Further evidence for a central role for protein kinase C in the activation of ERK following Ca2+ entry through CRAC channels was provided by acutely interfering with protein kinase C activity. The selective protein kinase C blocker G0–6983, which inhibits all known Ca2+-dependent conventional, novel, and atypical protein kinase C isoforms (46) , prevented thapsigargin (applied for 4 min) from activating ERK (Fig. 3 A; compare lanes 2 and 4, upper panel). Although G0–6983 had no effect on the extent of the cytoplasmic Ca2+ increase arising from store-operated Ca2+ entry (Fig. 3B ) or the extent of activation of ICRAC (Fig. 3C ), it suppressed arachidonic acid release (Fig. 3D ) and LTC4 secretion (Fig. 3E ), with an IC50 ~ 250 nM. Consistent with a major role for Ca2+-dependent protein kinase C, G0–6976, a protein kinase C inhibitor that is selective for the Ca2+-dependent isoforms but has no effect on Ca2+-independent ones (47) , significantly reduced ERK activation (Fig. 3A ) and suppressed arachidonic acid release as effectively as G0–6983 (Fig. 3D ).


Figure 3
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  		Figure 3. Protein kinase C inhibition prevents ERK phosphorylation, arachidonate release and LTC4 secretion. A) Western blot showing that thapsigargin-induced ERK phosphorylation in external Ca2+ is substantially reduced by pretreatment (15 min) with 2 µM G0–6976 or 2 µM G0–6983. Total protein loading (ERK2) is shown in the lower panel. B, C) Cytoplasmic Ca2+ signals due to store-operated Ca2+ entry (B) and the amplitude of ICRAC (C) are unaffected by pretreatment with G0–6983. B) Cells were exposed to thapsigargin in Ca2+-free solution for 10 min before 2 mM external Ca2+ was readmitted. n = 54 cells for control and 66 for G0–6983-treated cells in (B), and n = 5 for each condition in (C). D) Thapsigargin-evoked arachidonate release is suppressed by either G0–6983 or G0–6976 (both at 2 µM). E) G0–6983 impairs LTC4 secretion in a concentration-dependent manner.

Synergism between cytoplasmic calcium and protein kinase C
Direct stimulation of protein kinase C with phorbol ester (PMA) resulted in time-dependent ERK phosphorylation (Fig. 4 Aa) and to an extent similar to that seen following stimulation with thapsigargin for 4 or 8 min (Fig. 4Ab ). Despite activating ERK, PMA failed to promote arachidonic acid release (Fig. 4B , upper panel) or stimulate LTC4 secretion (Fig. 4B , lower panel). Importantly, PMA stimulation did not raise cytoplasmic Ca2+ (Fig. 4C ). Without the Ca2+ rise, cPLA2 would presumably be unable to translocate to intracellular membranes, a critical early step in enzyme activation. Hence, activation of ERK by protein kinase C is necessary but not sufficient for cPLA2 stimulation. Neither a Ca2+ rise in the absence of ERK phosphorylation (Figs. 1 and 2) or ERK phosphorylation without a Ca2+ rise (as seen with PMA, Fig. 4 ) are sufficient to activate cPLA2.


Figure 4
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  		Figure 4. Synergy between store-operated Ca2+ influx and protein kinase C. Aa) Western blots showing that stimulation with phorbol ester PMA (1 µM) for 5 or 10 min results in ERK phosphorylation (upper panel). Ab) Thapsigargin stimulation (4 or 8 min) induces similar levels of ERK phosphorylation (lower panel). B) PMA fails to evoke either arachidonic acid release (upper panel) or LTC4 secretion (lower panel). For comparative purposes, responses triggered by 4 min exposure to thapsigargin are included. C) Acute exposure to PMA fails to evoke a consistent cytoplasmic Ca2+ rise (n=51 cells). Ca2+ signals induced by thapsigargin on the same cell preparations are included (n=41 cells). D) Stimulation with thapsigargin (4 min) in different external Ca2+ concentrations results in a monotonic increase in arachidonate release (open circles). Pretreatment with 1 µM PMA for 10 min increases the amount of arachidionate released at lower external Ca2+ concentrations by thapsigargin (filled circles). E) Stimulation with thapsigargin for 4 min in 0.25 mM external Ca2+ evokes a modest increase in LTC4 secretion and this is significantly enhanced by PMA. Here, *denotes P < 0.05 and ** P < 0.01. F) PMA has little initial effect on the thapsigargin-evoked cytoplasmic Ca2+ signal in 0.25 mM external Ca2+.

Short-term potentiation driven by Ca2+ and protein kinase C
The dual requirement for a cytoplasmic Ca2+ rise and recruitment of the protein kinase C/MEK/ERK cascade raises the possibility of a facilitatory interaction between these major intracellular signaling pathways. To address this, we stimulated cells with thapsigargin, but in the presence of different external Ca2+ concentrations, in order to evoke different cytoplasmic Ca2+ rises. In low external Ca2+ (0.25–0.5 mM), 4 min stimulation with thapsigargin evoked a modest increase both in arachidonic acid release (Fig. 4D , open circles) and LTC4 secretion (Fig. 4E ). However, both responses were enhanced substantially when cells were preexposed to 1µM PMA (Fig. 4D , filled circles, and E). Importantly, protein kinase C had no potentiating effect when cells were stimulated with thapsigargin but in the absence of external Ca2+ (Fig. 4D ). Hence protein kinase C potentiates the activation of the ERK pathway following modest store-operated Ca2+ influx, thereby rendering small increases in cytoplasmic Ca2+ much more effective in driving arachidonic acid release. This is not due to a change in the cytoplasmic Ca2+ signal itself because the thapsigargin-evoked Ca2+ rise was unaffected by protein kinase C stimulation (Fig. 4F ). At longer stimulation times, the Ca2+ signal fell to a slightly greater extent in the presence of PMA than in its absence (~20% smaller after 10 min), consistent with the idea that protein kinase C may modulate CRAC channel activity, although to a lesser extent than reported previously using whole cell recording of ICRAC (48 , 49) .

Ca2+ entry through CRAC channels promotes 5-lipoxygenase translocation via ERK activation
Because arachidonic acid is a pleiotropic intracellular messenger (50) , we reasoned that its production must be tightly coordinated with its subsequent metabolism to LTC4. 5-Lipoxygenase activity is low in nonstimulated cells and therefore an increase in arachidonic acid per se would not translate into significant LTC4 production. In RBL cells, addition of arachidonic acid up to 20 µM led only to a small increase in leukotriene generation (51) . Consistent with this, we found that addition of 1–10 µM arachidonic acid to resting cells failed to result in synthesis of LTC4 (data not shown). 5-Lipoxygenase exists in distinct intracellular pools in resting RBL cells (52) but, on a rise in cytoplasmic Ca2+ concentration, translocates via its amino-terminal C2-like domain to the nuclear membrane (29 30 31) . We therefore investigated 5-lipoxygenase translocation using Western blotting of nuclear membrane extracts. Stimulation with thapsigargin resulted in a significant increase in 5-lipoxygenase bound to the nuclear membrane, but only when external Ca2+ was present (Fig. 5 A). This is due to translocation of 5-lipoxygenase from non-nuclear pools rather than de novo protein synthesis because thapsigargin (up to 30 min stimulation) does not change the overall amount of 5-lipoxygenase expressed (28) . The CRAC channel blocker 2-APB significantly reduced the translocation of the 5-lipoxygenase following stimulation with thapsigargin (Fig. 5B ; quantitative analysis shown in lower panel). Inhibition of ERK activation by U0126 can suppress 5-lipoxygenase translocation and subsequent enzyme activity (53 54 55 56 57) . Moreover, ERK has been found recently to phosphorylate 5-lipoxygenase directly and this is required for 5-lipoxygenase activation (58) . We therefore examined the effects of ERK activation on 5-lipoxygenase translocation in RBL cells. Inhibition of ERK with U0126 impaired translocation of 5-lipoxygenase (Fig. 5C ) and this reduction was highly significant (Fig. 5C , lower panel). Hence, ERK activation is required for translocation (and therefore stimulation) of the 5-lipoxygenase. ERK inhibition does not alter the Ca2+ rise following stimulation with thapsigargin (Fig. 1D ). Hence, a rise in Ca2+ alone is not sufficient for the translocation of 5-lipoxygenase in these cells. Although LTC4 secretion could be detected after 4 or 8 min stimulation with thapsigargin, we failed to convincingly observe translocation of the 5-lipoxygenase to nuclear membrane within 10 min of stimulation with thapsigargin (Fig. 5A ). This is consistent with numerous other studies that have also been unable to detect translocation to the nucleus shortly after stimulation despite an increase in 5-lipoxygenase activity (29 , 30) , Difficulty in observing 5-lipoxygenase translocation at early time points may therefore be due to translocation of only a small fraction of the total amount of enzyme in the cell, which is nevertheless effective as the stimulated enzyme has a relatively high affinity for substrate.


Figure 5
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  		Figure 5. 5-lipoxygenase translocates to the nuclear membrane following CRAC channel activation. A) Western blot showing 5-lipoxygenase binding to nuclear membrane for the different conditions indicated. B, C) 2-APB (B) and U0126 (C) suppress 5-lipoxygenase translocation to the nuclear membrane following thapsigargin stimulation. Typical gels are shown in the upper panels and quantitative analysis of several gels are depicted in the lower panels.

Antigenic stimulation evokes LTC4 secretion via recruitment of ERK in a Ca2+-dependent manner
To see whether Ca2+-dependent recruitment of ERK and subsequent stimulation of the 5-lipoxygenase enzyme occurred under more physiological conditions, we repeated the above experiments but now activated cell-surface FC{epsilon}RI (antigenic) receptors instead. These receptors increase inositol 1,4,5-trisphosphate levels via tyrosine kinase-dependent activation of phospholipase C{gamma}. Stimulation with antigen (1–5 µg/ml) resulted in Ca2+ release from the stores followed by Ca2+ influx into the cell (Fig. 6 A), through CRAC channels (32) . Although less effective than thapsigargin, stimulation in the presence, but not absence, of Ca2+ resulted in significant ERK activation (Fig. 6B ) as well as LTC4 secretion (Fig. 6C ). Importantly, U0126 and PD98059 suppressed LTC4 secretion in response to FC{epsilon}RI receptor activation (Fig. 6C ). Hence, like thapsigargin, Ca2+ influx following stimulation of FC{epsilon}RI receptors recruits the MEK/ERK pathway culminating in secretion of LTC4.


Figure 6
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  		Figure 6. Stimulation of antigenic receptors activates ERK and subsequent LTC4 secretion. A) Stimulation with antigen (DNP-BSA, 5 µg/ml) evokes a transient Ca2+ signal in the absence of external Ca2+, whereas a small, sustained plateau is seen when external Ca2+ is present. B) Stimulation with antigen results in ERK phosphorylation (upper gel; lower gel is loading control) and quantitative analysis from three gels is summarized in the histogram. C) Antigen stimulates LTC4 secretion and this is suppressed by preventing ERK activation with either U0126 or PD98059.

Ca2+ entry through CRAC channels activates c-fos expression
ERK can activate transcription of immediate genes like the nuclear transcription factor c-fos. To see whether Ca2+ influx through CRAC channels can stimulate c-fos transcription, we performed RT-PCR experiments using primers specific for c-fos. Stimulation with thapsigargin for just 4 min in the presence of external Ca2+ (followed by perfusion with Ca2+-free solution for 41 min) resulted in c-fos transcription (Fig. 7 A) and translation, as detected using Western blotting of nuclear extracts (Fig. 7B ). Transcription and translation of c-fos were prevented if cells were stimulated with thapsigargin either in the absence of external Ca2+ (Fig. 7A,B ) or in the presence of the CRAC channel blocker 2-APB (Fig. 7B ). Hence, Ca2+ influx through CRAC channels is required for thapsigargin-dependent c-fos expression. Intriguingly, we found that inhibition of ERK activation with either U0126 or PD98059 failed to reduce c-fos expression to any significant extent (Fig. 7C,D ; P>0.1 and 0.3, respectively). ERK does not contribute to linking CRAC channel activity to c-fos expression.


Figure 7
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  		Figure 7. c-fos expression following Ca2+ entry through CRAC channels. A) RT-PCR showing thapsigargin stimulation (4 min) in the presence of external Ca2+ results in c-fos gene expression but not in the absence of external Ca2+ or in the presence of 2-APB. Cells were stimulated with thapsigargin for 4 min and then perfused with Ca2+-free solution for 41 min prior to RNA extraction The upper panel shows control ß-actin loading. B) Western blotting of nuclear membrane shows that Ca2+ entry promotes c-fos protein expression and this is suppressed by 2-APB (30 µM; 5 min pretreatment). Same stimulation protocol as in (A). C) Upper panel depicts a typical nuclear membrane blot probing for c-fos expression in nonstimulated cells, and after stimulation with thapsigargin in the absence and then presence of U0126. Aggregate data from four gels is summarized in the histogram. D) As in (C) but now in the presence of PD98059.

DISCUSSION

The results of the present study demonstrate that Ca2+ entry through CRAC channels can (1) via recruitment of ERK, stimulate the production of the second messenger arachidonic acid through phosphorylation and subsequent activation of cPLA2 as well as drive secretion of the proinflammatory paracrine signal LTC4 through activation of the 5-lipoxygenase enzyme and (2) induce transcription and translation of the nuclear transcription factor c-fos. The former processes develop within minutes, whereas c-fos expression is manifest after tens of minutes and is maintained for at least 2 h (data not shown), considerably longer than the initial 4 min thapsigargin stimulation protocol we have followed. Hence, transient Ca2+ influx through CRAC channels can induce short- and long-term changes in cell function.

Activation of ERK following Ca2+ entry through CRAC channels is functionally important because it is required for cPLA2 activation and 5-lipoxygenase translocation, which generate intra- and intercellular messengers. Inhibition of ERK activation had no effect on the cytoplasmic Ca2+ rise that arose from the opening of the CRAC channels. Hence elevation of cytoplasmic Ca2+ is not sufficient to activate cPLA2 in the absence of ERK stimulation. Similarly, ERK activation in the absence of a Ca2+ rise (as occurs with PMA) also fails to activate cPLA2. Therefore, a rise in Ca2+ and activation of ERK are both needed for cPLA2 stimulation, at least in RBL cells. Whereas previous studies have considered these signals to function independently, our new results demonstrate that ERK activation is in fact Ca2+-dependent. Ca2+ entry through CRAC channels promotes robust translocation of the conventional Ca2+-dependent protein kinase C isozymes {alpha} and ßI to the cell periphery (presumably the plasma membrane), which is necessary for their activation. Chronic exposure to phorbol ester selectively down-regulates the Ca2+-dependent protein kinase C isozymes in RBL cells (45) and this greatly attenuates the ability of CRAC channel-mediated Ca2+ influx to stimulate cPLA2 and LTC4 secretion. Further evidence supporting a central role for Ca2+-dependent protein kinase C isozymes comes from studies using acute pharmacological block of these enzymes. Inhibition of protein kinase C results in suppression of ERK phosphorylation, cPLA2 activation and LTC4 secretion. Ca2+ entry through CRAC channels, therefore, promotes cPLA2 activation via two distinct pathways: 1) it promotes cPLA2 translocation to intracellular membranes via occupancy of the C2-like domain, and 2) it activates ERK through recruitment of Ca2+-dependent protein kinase C and MEK.

Strong synergy between cytoplasmic Ca2+ and ERK activation was seen following modest increases in cytoplasmic Ca2+ concentration. Such small Ca2+ elevations (as occurred following stimulation in 0.25 mM external Ca2+) evoked weak activation of cPLA2, but this was significantly enhanced by stimulation of protein kinase C. Such synergy is important because it enhances secretion of LTC4. Synergy between protein kinase C and Ca2+ could provide an explanation for previous reports in the literature documenting that the serine/threonine phosphatase inhibitor OA increases cPLA2 activity without altering cytoplasmic Ca2+ concentration (22) . By inhibiting phosphatase activity, OA would promote phosphorylation of cPLA2 such that basal Ca2+ might be sufficient to support enzyme activation. Even under these conditions though, cPLA2 activity was still dependent on the ambient Ca2+ levels because lowering cytoplasmic Ca2+ (by removing external Ca2+) reduced OA-induced cPLA2 activation (22) . Our findings can be nicely incorporated into the model proposed by Das and colleagues (23) . In this scheme, the Ca2+ rise enables the amino-terminal C2 domain to insert into the membrane but phosphorylation of Ser-505 is required for aliphatic amino acids at the cPLA2 active site rim to penetrate the membrane, thus positioning the active site for catalysis. Ca2+ drives membrane insertion as well as phosphorylation (via Ca2+-dependent protein kinase C recruitment of ERK). With small increases in cytoplasmic Ca2+ not only would a fraction of cPLA2 molecules insert into the membrane, but ERK activation would also be modest. Combined, this translates into weak arachidonic acid generation. If ERK is activated strongly by stimulating protein kinase C with phorbol ester, but leaving the cytoplasmic Ca2+ rise unaffected, more cPLA2 enzymes will be phosphorylated and therefore those that have inserted into the membrane will be catalytically more active. Larger rises in cytoplasmic Ca2+, as seen following stimulation with thapsigargin in 2 mM Ca2+, would be much more effective in recruiting both processes (membrane insertion and ERK stimulation), thus ensuring robust activation of cPLA2.

Generation of arachidonic acid following a cytoplasmic Ca2+ rise is not without problems. Like Ca2+, arachidonic acid is a promiscuous signal that can activate a wide range of intracellular processes (50) . Converting one promiscuous signal to another is unlikely to provide specificity. However, in addition to activating cPLA2, Ca2+ entry through CRAC channels promotes translocation of the arachidonic acid-metabolizing enzyme 5-lipoxygenase to the nuclear membrane. This is one of the main sites of arachidonic acid release by cPLA2, thus translocation of the 5-lipoxygenase ensures that as arachidonic acid is generated it will be metabolized to LTC4. We found that 5-lipoxygenase translocation was impaired by blocking ERK activation. Hence, ERK acts as a key co-ordinator/processor of Ca2+ influx. Following its activation on Ca2+ influx upon CRAC channels, ERK stimulates both cPLA2 and 5-lipoxygenase, thus ensuring rapid and tightly co-ordinated production of LTC4. In this way, ERK acts a versatile transducer of CRAC channel activity.

Ca2+ entry through CRAC channels is an important regulator of cell growth and proliferation (37) . In T cells, thapsigargin-evoked Ca2+ influx drives translocation of the Ca2+-dependent transcription factor NF-AT to the nucleus where it promotes increased transcription of the interleukin (IL)-2 gene and its receptor (59) . In the present report, we have found that Ca2+ influx during 4 min stimulation is sufficient for increased transcription and translation of the immediate early gene, c-fos. Importantly, gene activation was suppressed by the CRAC channel blocker 2-APB or by removing external Ca2+. Hence transient Ca2+ influx through CRAC channels is sufficient for gene expression. ERK activation can stimulate gene expression directly through phosphorylation of the transcription factor Elk-1 (60 , 61) , or indirectly via activation of either ribosomal protein S6 kinases (62) or mitogen- and stress-activated kinases (63) . However, inhibition of ERK activation with U0126 failed to suppress c-fos expression. Hence, CRAC channel-dependent activation of c-fos is not mediated by ERK to any appreciable extent. How Ca2+ entry through CRAC channels results in c-fos expression is unclear at present. Nevertheless, the results show that transient Ca2+ entry through CRAC channels can activate at least two distinct signaling pathways (ERK-dependent and -independent), which evoke temporally distinct cellular responses. The ERK-dependent pathway results in the production, within minutes, of arachidonic acid and LTC4, whereas an ERK-independent pathway contributes to c-fos expression.

Finally, cysteinyl leukotrienes like LTC4 have been recognized as playing a significant role in the pathophysiology of asthma and potent and effective cysteinyl leukotriene receptor antagonists have been developed for the treatment of this illness (64 65 66) . Our results revealing the signaling pathways that link CRAC channel activity to LTC4 secretion may help identify novel therapeutic targets for the development of drugs aimed at treating this growing disorder.

ACKNOWLEDGMENTS

This work was supported by an MRC Programme Grant to A.B.P. W-C.C is in receipt of an ORS studentship.

Received for publication February 28, 2006. Accepted for publication June 2, 2006.

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