Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
* Author for correspondence (e-mail: hirashim{at}phar.nagoya-cu.ac.jp)
Accepted 18 February 2005
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Summary |
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Key words: Mast cell, Complexin, Exocytosis, SNARE, Allergy, Rat
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Introduction |
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In the present study, we examined the expression and role of complexin in mast cells, which are typical non-neuronal secretory cells. In mast cells, cross-linking of high-affinity receptors for IgE (FcRI) by multivalent antigen activates an intracellular signaling cascade and leads to the exocytotic release of granular contents (degranulation), resulting in allergic responses (Abraham and Malaviya, 1997
; Swann et al., 1998
; Turner and Kinet, 1999
; Kinet, 1999
). We have previously studied the mechanism of degranulation in mast cells and identified the protein and lipid molecules that regulate this process (Hibi et al., 2000
; Kato et al., 2002
; Kato et al., 2003
). We and other groups have reported that SNARE proteins are involved in degranulation in mast cells (Guo et al., 1998
; Hibi et al., 2000
; Paumet et al., 2000
; Blank et al., 2001
; Blank and Rivera, 2004
), although the active isoforms in mast cells are different from those in neuronal cells. However, there is little information available on the proteins that regulate SNARE proteins in mast cells, except for Munc18-2 (Martin-Verdeaux et al., 2003
).
In this study, we found that complexin II was expressed in mast cells. In experiments using complexin II knockdown cells, we found that complexin II positively regulated exocytotic release in mast cells. Immunocytochemical experiments revealed that complexin II changed its localization from the cytoplasm to the plasma membrane, and this translocation occurred in the absence of extracellular Ca2+, while degranulation was inhibited almost completely. We also found that the degranulation induced by phorbol 12-myristate 13-acetate (PMA) and A23187 depended on extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although the association of complexin II with SNARE complex is not sufficient to trigger exocytotic membrane fusion.
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Materials and Methods |
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Cell culture
A mast cell line, rat basophilic leukemia cell (RBL-2H3), was cultured in Eagle's minimal essential medium from Nissui (Tokyo, Japan) with 10% fetal calf serum (Boehringer Mannheim) at 37°C in an atmosphere of 5% CO2. Mastocytoma P815 was cultured in RPMI1640 (Gibco) supplemented with 10% fetal calf serum at 37°C in an atmosphere of 5% CO2.
RT-PCR
Poly(A)+ RNA was obtained with a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech) from 1x 107 cells of RBL-2H3 and cerebrum of Sprague-Dawley rat (6 weeks), and served as a template for cDNA synthesis with SuperScript II RT (Gibco BRL), as reported previously (Hibi et al., 2000). The primer pairs used to amplify complexins I and II were 5'-ATGGAGTTCGTGATGAAACAAG-3' (sense)/5'-TTACTTCTTGAACATGTCCTGCA-3' (anti-sense) and 5'-ATGGACTTCGTCATGAAGCA (sense)/5'-TTACTTCTTGAACATGTCCTGCA-3' (anti-sense), respectively. PCR products were extracted from agarose gel with Gene Clean (Bio 101) and subcloned into the TA cloning vector pCRII (Invitrogen). Cloned PCR products were sequenced with a DSQ1000 DNA sequencer (Shimadzu, Kyoto, Japan) using FITC-labeled M13 universal primer.
Western blotting
RBL-2H3 or P815 cells (5x 106) were lysed with lysis buffer (10 mmol/l HEPES, 1% Triton-X100, 1 mmol/l EDTA, 50 mmol/l NaF, 2.5 mmol/l p-nitrophenyl phosphate, 1 mmol/l Na3VO4, 1 mmol/l PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin). After centrifugation at 23,000 g for 20 minutes, supernatant was mixed with an equal volume of Laemmli sample buffer and boiled for 5 minutes. For cellular lysate of rat brain, rat cerebrum lysate was purchased from Transduction Laboratories. Samples were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. After blocking with phosphate buffer containing 5% skimmed milk, blots were probed with primary antibody for 1 hour. As primary antibodies, anti-complexin I antibody (dilution 1:200; Santa Cruz Biotechnology), anti-complexin II antibody (dilution 1:500; Transduction Laboratories), anti-syntaxin-3 antibody (dilution 1:250; Alomone Labs, Israel), anti-syntaxin-4 antibody (dilution 1:500; Santa Cruz Biotechnology), anti-synaptotagmin II antibody (dilution 1:200 Santa Cruz Biotechnology), and ß-actin antibody (dilution 1:4000; Sigma) were used. After being washed with 0.1% Tween 20 in PBS, membrane was treated with anti-mouse IgG conjugated with horseradish peroxidase. Immunoreactivity was detected by enhanced chemiluminescence (ECL, Amersham Pharmacia) with a LAS-1000 (FUJI FILM, Tokyo Japan) and analyzed by Image Gauge (FUJI FILM).
Plasmid construction and transfection
Poly(A)+ RNA was obtained as described above. For the knockdown of complexin II, 5'-GGATCCATGGACTTCGTCATGAAGCA-3' (sense; BamHI site is underlined)/5'-GCGGCCGCTTACTTCTTGAACATGTCCTGCA-3' (anti-sense; NotI site is underlined) was used as a primer pair. For the expression of myc-tagged complexin II, 5'-GGATCCATGGACTTCGTCATGAAGCA-3' (sense; BamHI site is underlined)/5'-GAATTCTTACTTCTTGAACATGTCCTGCA-3' (anti-sense; EcoRI site is underlined) was used as a primer pair. PCR products were extracted from agarose gel with Gene Clean (Bio 101) and subcloned into the TA cloning vector pCRII. Cloned PCR products were sequenced with a DSQ1000 DNA sequencer. Verified cDNA was ligated to pcDNA3 (Invitrogen) in the antisense direction to knockdown the expression of complexin II. For the expression of myc-tagged complexin II, cDNA was ligated to pCMV-Tag5 (Stratagene). RBL-2H3 cells (5x 105 cells/500 µl) were electroporated in cold PBS with 40 µg of plasmid DNA at 250 V and 950 µF using Gene Pulser II (Bio-Rad) (Hibi et al., 2000). Stable clones with reduced expression of complexin II were selected by G418 (500 µg/ml) and western blotting with anti-complexin II or anti-myc antibody.
Assay of secreted ß-hexosaminidase
Degranulation of RBL-2H3 cells was monitored by measuring the activity of a granule-stored enzyme, ß-hexosaminidase, secreted in cell supernatant (Amano et al., 2001). Cells were sensitized by anti-DNP IgE for 30 minutes and incubated with an average of six DNP groups conjugated with BSA (DNP6-BSA) for 30 minutes at 37°C. Aliquots of supernatant were incubated with substrate solution (2 mmol/l p-nitrophenyl-N-acetyl-ß-D-glucosaminide in 100 mmol/l citrate, pH 4.5) for 1 hour at 37°C. After the reaction was terminated with Na2CO3-NaHCO3 buffer, absorbance at 405 nm was measured. Release activity relative to the total ß-hexosaminidase content of the cells was calculated. Total ß-hexosaminidase content was determined by dissolving cells with 0.1% Triton-X100.
Intracellular Ca2+ measurement
RBL-2H3 cells were loaded with Fura2/AM (Molecular Probes; Eugene, OR) for 30 minutes at 37°C and washed twice with HEPES-buffered saline (140 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 0.6 mmol/l MgCl2, 0.1% glucose, 0.1% BSA, 0.1 mg/ml sulfinpyrazone and 10 mmol/l HEPES, pH 7.4). For experiments with different extracellular Ca2+ concentration, concentration of CaCl2 in HEPES-buffered saline was varied from 0 to 2 mmol/l. Cells were sensitized with anti-DNP IgE for 30 minutes and stimulated with DNP6-BSA (100 ng/ml). For the stimulation with Ca2+ ionophore and phorbol ester, A23187 (1 µmol/l) and PMA (50 ng/ml) were used instead of DNP6-BSA. The fluorescence intensities with excitation at 340 and 360 nm were measured, and the ratio (F340/F360) and Ca2+ concentration were calculated by a spectrofluorometer linked to a personal computer (RF-5300PC; Shimadzu, Japan), following a procedure described previously (Grynkiewicz et al., 1985). During measurement of the intracellular Ca2+ concentration, cells were kept at 37°C.
Immunocytochemistry
Cells (1x 105) were plated in a ZOG-3 glass-bottom chamber (Elekon Science; Chiba, Japan) and incubated for 18 hours. After cells were washed with PBS, they were fixed with PBS containing 4% paraformaldehyde and permeabilized with PBS containing 0.2% Triton-X100. After blocking with 5% BSA in PBS, cells were incubated with anti-complexin II antibody (dilution 1:100; Transduction Laboratories) or anti-myc antibody (9E10, dilution 1:50; Santa Cruz Biotechnology) at 4°C overnight. Cells were treated with fluorescence-labeled secondary antibody (FITC-conjugated anti-mouse IgG) after they were washed three times with PBS for 10 minutes. Fluorescent images were taken with a confocal laser scanning microscope (Zeiss, LSM-510) with a 63x objective lens (Plan-Apochromat 63x/1.4 oil). Samples were excited at 488 nm with an Ar laser and fluorescence was observed with an LP560 filter (Kato et al., 2002).
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Results |
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Characterization of complexin II-knockdown cell
To investigate the role of complexin II in exocytotic release in mast cells, we obtained RBL-2H3 cells that expressed a low level of complexin II. After selection with G418, four independent clones with a low expression of complexin II (knockdown cells) were picked up by western blotting analysis. All these clones expressed complexin II at less than 50% of the level in wild-type cells, and they all exhibited similar behaviors in this study. Fig. 2A shows western blotting of cell lysate derived from four knockdown clones. Expression levels of complexin II in knockdown clones (kd-1, 2, 3 and 4) were 42, 46, 40 and 38% of those in wild-type cells, respectively. The expression levels of syntaxin-3 and syntaxin-4, which are SNARE proteins expressed in RBL-2H3 cells and thought to be involved in degranulation, were not significantly changed in knockdown cells (Fig. 2A). In addition, the expression of synaptotagmin II, which is the most abundant isoform of synaptotagmin and regulates exocytotic release in RBL (Baram et al., 1999), was not affected (Fig. 2A). The expression of complexin II was attenuated in knockdown cells, but its intracellular distribution was not changed, and complexin II was distributed throughout the cytoplasm and the nucleus, as observed in wild-type cells (Fig. 2B).
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The exocytotic release of complexin knockdown cells was investigated by quantifying ß-hexosaminidase secreted in medium. Fig. 3A shows the timecourse of degranulation induced by antigen stimulation. A significant inhibition of degranulation was observed in complexin II knockdown cells. There was no difference in the total amount of ß-hexosaminidase between wild-type and knockdown cells.
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Translocation of complexin II after stimulation
To elucidate the mechanism by which complexin II regulates exocytosis in mast cells, the intracellular localization of complexin II before and after antigen stimulation was observed. Before stimulation, complexin II was distributed throughout the cell as described in Fig. 1. However, upon stimulation, the distribution of complexin II changed dramatically, and it was translocated to the plasma membrane (left images in Fig. 4A,B). This translocation to the plasma membrane was clear at about 3 minutes after stimulation. The distribution of fluorescence signal became more punctate. Similar results were obtained in RBL-2H3 cells expressing myc-tagged complexin II with anti-myc antibody (right images in Fig. 4A,B). Next, we investigated this translocation in the absence of extracellular Ca2+. Translocation to the plasma membrane was observed even in the absence of extracellular Ca2+ (Fig. 4C). Under this condition, exocytotic release was inhibited almost completely (Fig. 4D). Stimulation with thapsigargin in the absence of extracellular Ca2+ gave similar results (Fig. 4E,F). These results suggest that the transient elevation of the intracellular Ca2+ concentration is sufficient for translocation to the plasma membrane, and neither sustained Ca2+ increase due to the influx of extracellular Ca2+ nor the activation of IgE receptors was necessary.
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Ca-dependence of degranulation on extracellular calcium concentration
Experiments with complexin-knockout mice (Reim et al., 2001) suggested that complexin is involved in the calcium-dependence of exocytotic release in neuronal cells. Therefore, we compared the Ca2+-dependence of degranulation in wild-type and knockdown cells. Cells were stimulated with the calcium ionophore A23187 and PMA. The calcium ion concentration in extracellular solution was changed from nominally free to 2 mmol/l. In Fig. 5, the Ca2+-dependent component of degranulation activity, which was calculated by subtracting the value of degranulation at 0 mmol/l, is shown. In both wild-type and knockdown cells, degranulation decreased as the extracellular Ca2+ concentration decreased, but the dose-response curve of knockdown cells was shifted toward a higher concentration of Ca2+ (Fig. 5A). To compare the Ca2+ sensitivity of Ca2+-dependent degranulation between wild-type and knockdown cells, degranulation activity shown in Fig. 5A was re-plotted against intracellular Ca2+ concentration (Fig. 5B). As shown in an inset of Figure 5B, intracellular Ca2+ concentration reached a plateau at about 400 seconds after stimulation. The timecourse of intracellular Ca2+ concentration change induced by PMA and A23187 did not differ between wild-type and knockdown cells. Therefore, we considered an average concentration from 400 seconds to 20 minutes after stimulation as representative of intracellular Ca2+ concentration, and estimated the intracellular Ca2+ concentrations when cells were stimulated in various extracellular Ca2+ concentrations. Using these estimated intracellular Ca2+ concentrations, we re-plotted Ca2+ dependency of degranulation (Fig. 5B). Data were fitted with the Hill equation:
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Discussion |
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In experiments using complexin II knockdown cells, we found that complexin II positively regulated exocytotic release. These inhibitory effects were not due to inhibition of the expression of syntaxin (Fig. 2A) or antigen-induced Ca2+ mobilization (Fig. 2D), which suggests that complexin II plays a role in the process of membrane fusion between secretory granules and the plasma membrane.
To investigate the mechanism of the regulation induced by complexin II, we observed its intracellular distribution after antigen stimulation. Before stimulation, complexin II resided in the cytoplasm; however, it was translocated to the plasma membrane after stimulation (Fig. 4B). This translocation was observed at 3 minutes after stimulation and degranulation also became clear in the same time window, suggesting that this translocation is closely related to degranulation. As complexin selectively associates with the ternary SNARE complex, but not with monomeric SNARE proteins, this translocation might be due to the association of complexin II with SNARE complex on the plasma membrane that is formed by stimulation. The distribution of fluorescence signal became more punctate, and clear spots appeared on the plasma membrane after stimulation. This spot-like structure might reflect the sites of exocytotic membrane fusion. Interestingly, the translocation to the plasma membrane occurred even in the absence of extracellular Ca2+ (Fig. 4C). Furthermore, since stimulation by thapsigargin in the absence of extracellular Ca2+ caused the translocation of complexin II (Fig. 4E), a transient increase in intracellular Ca2+ is sufficient for translocation, and a signal through the IgE receptor is not required. When cells were stimulated without extracellular Ca2+, degranulation was inhibited almost completely (Fig. 4D,F). This suggests that the translocation to the plasma membrane of complexin II is not enough to induce membrane fusion between secretory granules and the plasma membrane, and Ca2+ influx from the extracellular medium triggers membrane fusion. Transient increase in Ca2+ might be necessary to form a ternary SNARE complex, to which complexin II binds. Once the SNARE complex is formed, complexin II can associate with the SNARE complex, and Ca2+ influx from the extracellular medium is not required. This notion is consistent with the model that the Ca2+-independent association of complexin with the SNARE complex puts the complex in a metastable state, which is essential for efficient, fast Ca2+-dependent neurotransmitter release (Chen et al., 2002; Marz and Hanson, 2002
). These observations, that complexin II was translocated to the plasma membrane but translocation itself did not trigger degranulation, were observed for the first time and provide useful information for understanding the mechanism of exocytosis regulation, not only in mast cells but also in neuronal cells.
Interestingly, knockdown of complexin II affected the Ca2+ sensitivity of degranulation (Fig. 5). While maximum release was not significantly changed, Kd and the Hill coefficient suggested that the Ca2+ sensitivity was clearly reduced in knockdown cells. A similar reduction of Ca2+ sensitivity was observed by Reim et al. (Reim et al., 2001) in excitatory postsynaptic currents (EPSC) of hippocampal neurons from complexin knockout mice. They investigated the Ca2+ dependence of synchronous evoked transmitter release and estimated the Kd value for extracellular Ca2+ concentration using the Hill equation. They found that the Kd of double knockout mice for complexins I and II increased twice as much as that of single knockout mice for complexin II.
As we could not obtain data at higher concentrations of intracellular Ca2+, due to cellular damage caused by long exposure to high concentrations of Ca2+, the dose-response curve for knockdown clones does not seem to be saturated (Fig. 5B). Nonlinear regression analysis allows us to estimate values related to intracellular Ca2+ dependency, which is extremely important for understanding the mechanism of calcium-dependent secretion, although lack of measured values at saturated phase did not provide an ideal condition for curve-fitting using the Hill equation.
Complexin itself does not have an apparent binding site with Ca2+ and its binding to the SNARE complex is not affected by Ca2+ (Pabst et al., 2000). Therefore, it is unlikely that complexin II is the Ca2+ sensor that regulates Ca2+-dependent exocytosis. As complexin II is translocated to the plasma membrane after stimulation, there are at least three steps that may be sensitive to the Ca2+ concentration: the translocation of complexin II to the plasma membrane; the association of complexin II with the SNARE complex on the plasma membrane; and the induction of exocytotic membrane fusion. Considering the finding that exocytotic release requires a sustained increase in the intracellular Ca2+ concentration due to Ca2+ influx, it is probable that the latter two steps have major contributions to the Ca2+-sensitivity of degranulation induced by ionophore and PMA. Change in not only Kd but also the Hill coefficient suggests that complexin II regulates the conformation of Ca2+-binding sites of the Ca2+ sensor. So far, synaptotagmin is the most likely candidate for the Ca2+ sensor. As synaptotagmin has two Ca2+-binding sites, complexin II might regulate the conformation or binding ability of these sites. In mast cells, synaptotagmins II, III, V and IX are expressed (Baram et al., 2001
), and Baram et al. (Baram et al., 1999
) reported that synaptotagmin II, which is a major isoform of synaptotagmin, negatively regulates Ca2+-dependent exocytosis in RBL-2H3 cells. As the expression level of synaptotagmin II was not affected in knockdown cells, as shown in Fig. 2A, a study on the interaction of synaptotagmin II with the SNARE complex in the presence of complexin II should shed light on the mechanism of the reduction of Ca2+ sensitivity in complexin II knockdown mast cells.
Pabst et al. (Pabst et al., 2000) investigated the association of complexin with the SNARE complex using different isoforms of syntaxin. They showed that complexin binds SNARE complexes comprised of SNAP-25, VAMP 2 and syntaxin 3 as efficiently as it binds with complexes comprised of SNAP-25, VAMP 2 and syntaxin 1. However, complexin did not bind to the SNARE complex containing syntaxin 4. This finding, together with the result that complexin is involved in degranulation, suggests that syntaxin 3 plays a major role as t-SNARE in mast cells. This is also supported by the observation that Munc18-2, which associates with syntaxin 3 but not syntaxin 4, is involved in degranulation in mast cells (Martin-Verdeaux et al., 2003
).
Very recently, Abderrahmani et al. (Abderrahmani et al., 2004) showed that complexin I, but not complexin II, regulates secretion in pancreatic ß cells. They also knocked down the expression of complexin I and showed a reduction in secretion induced by nutrients. As the SNARE complex formed by syntaxin I, VAMP and SNAP25 is required for exocytotic release in pancreatic ß cells (Wheeler et al., 1996
; Regazzi et al., 1996
), complexin I serves as a positive regulator of SNARE-like complexin II in mast cells, although different isoforms of SNARE and complexin act in these cells. The involvement of complexin in degranulation provides an insight into the mechanism of allergy and the development of anti-allergy drugs.
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