|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Proteomic analysis to identify early molecular targets of pregabalin in C6 glial cells
Seyeon Park*1 and Joomin Lee†
*Department of Applied Chemistry, Dongduk Women's University, Seoul 136-714, Korea, and †Department of Food and Nutrition, Yonsei University, Seoul 134, Korea
Pregabalin is a lipophilic amino acid derivative of γ-amino butyric acid that displays anticonvulsant and analgesic activities against neuropathic pain. Although a role for glial cells as an important player in pain control and also as a new target for pain medicine has been suggested, the effect of pregabalin on glial cells has not been elucidated. In the present study, we have investigated the action of pregabalin on the glial cell proteome. To identify immediate early protein targets of pregabalin in glial cells, a differential proteomics approach in C6 rat glioma cells treated with pregabalin was used. Seven proteins that sensitively reacted to pregabalin treatment were identified using two-dimensional gel electrophoresis and MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS). The calcium-ion-binding chaperone, calreticulin, and the oxidative response protein, DJ-1, were up-regulated after pregabalin treatment. Hsp (heat-shock protein)-90-β, cytoskeleton protein actin and myosin also showed quantitative expression profile differences. Functionally relevant to the proteome result, immediate actin depolymerization was observed after treatment with pregabalin. These results suggest a previously undefined role of pregabalin in the regulation of chaperone activity and cytoskeleton remodelling in glial cells.
Key words: C6 glial cell, calreticulin, cytoskeleton, DJ-1, pregabalin, proteomic approach
Abbreviations: Epac, exchange protein directly activated by cAMP, GABA, γ-amino butyric acid, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, Hsp, heat-shock protein, MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS, PBST, PBS containing 0.1% Tween 20
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Pregabalin and gabapentin are lipophilic structural analogues of GABA (γ-amino butyric acid) that were developed as potential anticonvulsants (Bryans et al., 1999). Many studies have shown that pregabalin and gabapentin do not act as GABA agonists and, indeed, have little demonstrable effect on any aspect of GABA transmission (Field et al., 1997; Houghton et al., 1998; Taylor et al., 1998; Maneuf et al., 2003). It is still not certain what the important mechanisms are underlying their anticonvulsant effect. A previous study has added clarity to the current understanding of pharmacology by concluding that these drugs are not an inhibitor of any conventional subtype of voltage-gated calcium channel, but rather selectively block calcium channels that contain the α2δ-1 subunit, with pharmacodynamic effects and a cellular specificity which mirrors the presence, structure and biochemical state of the α2δ-1 protein (Brown et al., 2005). Blockade of voltage-gated calcium channels containing the α2δ-1 subunit is believed to be the predominant pharmacological mechanism of both gabapentin and pregabalin (Sills, 2006). Pregabalin reduces nociceptive behaviours in animal models of neuropathic pain or inflammation, such as nerve ligation, injection of immune antigens, herpes infection, arthritis, diabetes, postoperative pain and thermal injury (Xiao et al., 1996; Field et al., 1997; Houghton et al., 1998; Partridge et al., 1998; Taylor et al., 1998; Chen et al., 2001; Takasaki et al., 2001). However, the mechanism by which pregabalin initiates cellular change toward physiological effects has not been elucidated. To understand the molecular basis of pregabalin-induced changes in an intracellular milieu, it is important to identify the expression or function of proteins modified by pregabalin. Little is known about the protein products produced by glial cells exposed to pregabalin since most studies have focused on the physiological effect of pregabalin.
Microglia cells are rarely dividing in the intact spinal cord (Horner et al., 2000). However, microglia proliferation occurs after nerve injury and was found in diverse models of neuropathic pain (Graeber et al., 1988; Suter et al., 2007). Glia activation in the spinal cord after nerve injury was reported to be associated with pain behaviour (Garrison et al., 1991). Later, it was found that spinal injection of a glial inhibitor fluorocitrate can reduce hyperalgesia (Meller et al., 1994). Glial activation has been studied in different animal models, such as neuropathic pain after injury to peripheral nerves or the spinal cord, inflammatory pain after injection of inflammatory substances (e.g. formalin) into a hindlimb, cancer pain after inoculation of tumour cells or orofacial pain after lesion of joint muscle (Colburn et al., 1997; Fu et al., 1999; Honore et al., 2000; Hains et al., 2006; Sessle et al., 2007). Both microglia and astrocytes are activated in these pain models and interact with neurons in complex pain pathophysiology (Suter et al., 2007). The inflammatory mediators released by activated glial cells, such as TNF-α (tumour necrosis factor-α) and IL (interleukin)-1β not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions (Suter et al., 2007). Pain can also be potentiated by growth factors such as BDNF (brain-derived neurotrophic factor) and bFGF (basic fibroblast growth factor) that are produced by glia to protect neurons. Thus glial cells can control pain when they proliferate and are activated to produce various pain mediators. That is why, in the present study, we focused on the glial cell model to elucidate molecular mechanisms of neuronal-specific anticonvulsants.
In the present study, we have analysed protein targets aimed at pregabalin in glial cells using a proteomic approach. This methodology provides important qualitative information on post-translational modifications to each protein and quantitative data on protein expression in response to a particular stimulus. Furthermore, our aim was to identify early mechanisms whereby changes in the intracellular milieu induce a molecular response in the glial cell line. Thus the cells were treated within 2 h. This is particularly important because it provides data on early cellular events, such as the stimulus and signalling cascades triggered. According to pharmacokinetic studies, the maximum plasma concentration of gabapentin after administration of a single 400 mg neurontin tablet to 12 volunteers was 3.33±1.19 μg/ml (Bahrami et al., 2006; Jalalizadeh et al., 2007).
2. Materials and methods
2.1. Cell culture and treatment
The rat glioma cell line C6, purchased from Korean Cell Line Bank, was maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS (fetal bovine serum) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). The cells were incubated in 5% CO2 in an air-humidified incubator at 37°C. The cells were treated with gabapentin or pregabalin in DMSO (not exceeding 0.01%, an equal volume of DMSO was used as a vehicle for control) at the concentrations indicated. Gabapentin was purchased from Sigma and pregabalin was provided by Pfizer Pharmaceutical Korea.
2.2. Preparation of total protein extracts
To obtain total protein extracts, 5×107 cells were resuspended in a total volume of 500 μl of buffer containing 8 M urea, 2 mM tributylphosphine, 4% (w/v) CHAPS, 0.2% carrier ampholyte (pH 4–7; Bio-Rad) and a protease inhibitor cocktail (Sigma–Aldrich). Cells were disrupted by ten strokes with a Model XL-2020 sonicator (Misonix). After 1 h incubation at room temperature (25°C), cell lysates were centrifuged at 12000 g for 1 h at 13°C. The supernatant was collected and the protein concentration was determined using the Bio-Rad detergent compatible protein assay.
2.3. Proteome analysis
2.3.1. Two-dimensional PAGE and gel analysis
Proteins (1.5 mg) were hydrated overnight in 8 M urea, 4% CHAPS, 0.2% carrier ampholyte and 0.0002% Bromophenol Blue at 4°C and applied to isoelectric focusing gel strips (pH 4–7 linear, 17 cm in length; Bio-Rad). The gel strips were rehydrated overnight in 8 M urea, 4% CHAPS, 0.2% carrier ampholyte and 0.0002% Bromophenol Blue at 20°C. Isoelectric focusing was initiated at 100 V for 2 h, 250 V for 2 h and gradually increased to 10000 V for 10 h. The focusing process was carried out for 10000 Vh and held at 500 V for 24 h. After isoelectric focusing, IPG strips were equilibrated with 0.05 M Tris/HCl (pH 8.8), 0.6 mM TBP (tributyl phosphate), 5% iodoacetamide, 6 M urea, 20% glycerol and 2% SDS for 20 min. Strips were then transferred on to vertical slab SDS/PAGE (12% gels) and sealed with 1% low-melting-point agarose. SDS/PAGE was run for 30 min at a constant current of 16 mA per gel, and at 24 mA/gel at 15°C. After electrophoresis, the proteins were transferred on to an Immobilon-P PVDF membrane (Millipore) or visualized by Coomassie Brilliant Blue G-250 staining of the gels. Images were digitized using a Model GS-800 calibrated densitometer (Bio-Rad) and analysed by PDQuest two-dimensional analysis software (Bio-Rad).
2.3.2. Evaluation of differentially represented spots
Quantitative differences were determined only when a matched spot was found to show the same degree of down- or up-regulation in duplicate experiments. Matching spots in gels from the same sample were identified and their intensities were measured using an Image Master two-dimensional system. Analysis was performed on approx. 200 different protein spots per sample. For each spot, the intensity value in a pregabalin-treated gel was divided by its intensity value obtained in the control gel. The logs of these ratios were calculated (termed LR). LR means and median values were clustered around the zero value, which was expected if errors associated with the analysis were random and normally distributed. Spots showing an expression 5-fold less or greater than the control were considered statistically significant, differentially expressed protein species.
2.3.3. In-gel enzymatic digestion and MS
In-gel digestion was performed mainly as previously described (Rosenfeld et al., 1992). Spots were excised from the stained gel, destained with 0.1 M ammonium bicarbonate/50% acetonitrile (Sigma–Aldrich) and dried with a Speed Vac plus SC1 10 (Savant). The gel was rehydrated in a solution containing 1 M DTT (dithiothreitol) and 0.1 M ammonium bicarbonate (pH 7.8) for 30 min at 56°C. After a subsequent incubation in a solution containing 1% iodoacetamide and 0.1 M ammonium bicarbonate (pH 7.8) for 30 min in the dark, the gel was washed with 0.1 M ammonium bicarbonate/50% acetonitrile and dried using the Speed Vac apparatus. The gel was rehydrated in a trypsin solution (Promega). After incubation overnight at 37°C, peptides were sonicated for 30 min. Digested samples were removed and subjected to a desalting/concentration step on a μZipTipC18 column (Millipore) using acetonitrile as an eluent before MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) analysis. Peptide mixtures were loaded on to the MALDI system using the dried-droplet technique and α-cyano-4-hydroxycinnamic acid (Sigma–Aldrich) as the matrix, and were analysed using a Voyager-DE-PRO mass spectrometer (Applied Biosystems). Internal mass calibration was performed using peptides derived from enzyme autoproteolysis. The Dataexplorer software package was used to identify spots from MS-Fit (Protein Prospector; http://prospector.ucsf.edu) and the Mascot server (Matrix Science, London, U.K.) by mass searching of rat sequences. Candidates identified by peptide-mapping analysis were evaluated further by comparing their calculated mass and isoelectric points using the experimental values obtained by two-dimensional gel electrophoresis.
2.4. Western blot analysis
For Western blot analysis, amounts of protein extracts were obtained from C6 cells with or without pregabalin and analysed by SDS/PAGE (10% gels). Then, proteins were transferred on to nitrocellulose membranes (Schleicher and Schuell). After transfer, membranes were saturated by incubation at 4°C overnight with 10% (w/v) non-fat dried skimmed milk in PBST (PBS containing 0.1% Tween 20) and incubated with the indicated antibody (Cell Signaling) for 3 h. After three washes with PBST, membranes were incubated with an anti-rabbit immunoglobulin coupled with peroxidase (Santa Cruz Biotechnology). After 60 min of incubation at room temperature, the membranes were washed three times with PBST and the blots were developed using an enhanced chemiluminescence kit (Amersham Biosciences). Normalizations were performed with the polyclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (Santa Cruz Biotechnology). Blots were quantified using a Gel Doc 2000 densitometer (Bio-Rad).
2.5. Fluorescence dye staining
The cultures were stained for the detection of F-actin formation with phalloidin–Alexa Fluor® 568 (Molecular Probes). For the F-actin staining, the fixed cells were permeabilized for 3 min at room temperature in PBS supplemented with 0.05% Triton X-100. After washing with PBS, the cultures were treated with 5 units/ml phalloidin–Alexa Fluor® 568 and imaged under a fluorescence microscope with a WG filter set equipped with a Photometrics CoolSNAP camera (Roper Scientific).
2.6. Statistical analysis
Statistical analysis was performed using MINITAB. *Indicates P<0.05 compared with the control.
3.1. Changes in the C6 proteome profile after the treatment with pregabalin
Approx. 200 different spots that were focused in the pH 4–7 range were analysed per sample. After exposing cells to pregabalin, four intensified spots and two attenuated spots became apparent (Figures 1 and 2). Each of these spots was excised, digested with trypsin and analysed using MALDI–TOF-MS. Peptide mass fingerprint analysis and non-redundant sequence database matching allowed the unambiguous identification of all of the analysed species. Figure 1 and Table 1 detail the nature of each identified spot, its two-dimensional gel electrophoresis co-ordinates and relative sequence coverage.
Table 1 List of spots/proteins sensitive to pregabalin treatment in C6 cells, detected by two-dimensional gel electrophoresis and identified by peptide mass fingerprint analysis
The identified proteins were the only proteins detected in a given spot. The spot numbers are the same as those in Figure 1.
Several protein spots were members of the chaperone family. These spots included, for example, proteasome endopeptidase complex δ chain, Hsp (heat-shock protein) and calreticulin. DJ-1 also displays chaperone activity (Shendelman et al., 2004). Some proteins, including actin and myosin, belong to components of the cytoskeletal proteins.
3.2. Identification of calreticulin and DJ-1
MALDI–TOF-MS and quantitative analysis revealed that the protein spot with a sequence coverage range of 34% of the total amino acid sequences of calreticulin was a precursor form that included the leader sequence, comprising amino acids 1–17 (Figure 3).
We carried out Western blot analysis of lysates obtained from C6 cells treated with 50 μM pregabalin to confirm the proteome result. Treatment of C6 cells with 50 μM pregabalin for 1 and 2 h up-regulated calreticulin (P<0.05 at 1 h and P<0.01 at 2 h; Figure 4A); this effect was concentration-dependent (Figure 4). Similarly, 50 μM gabapentin up-regulated calreticulin (Figure 4A). Up-regulation of DJ-1 protein was also confirmed by Western blot analysis (P<0.05; Figure 5). Gabapentin also induced the up-regulation of DJ-1 protein.
3.3. The effect of pregabalin on actin polymer
Differential regulation of actin by pregabalin was explored to determine whether pregabalin affected its polymerization in glial cells. Cultures were supplemented with pregabalin (50 μM) for 1 h. Sparse web-like F-actin filaments were formed around the cell bodies in a larger number of glial cells treated with pregabalin than in the control (Figure 6).
Glia cells can control pain when they are activated to produce various pain mediators (Honore et al., 2000; Hains et al., 2006; Sessle et al., 2007; Suter et al., 2007). Thus we focused on the glial cell model to explain molecular mechanisms of the neuronal-specific anticonvulsant, pregabalin. Differential expression of chaperone protein and cytoskeleton proteins in pregabalin-treated glial cells was demonstrated using proteomics with MALDI–TOF-MS. Functionally relevant actin depolymerization was also observed after treatment with pregabalin. Taken together, these results suggest an undefined role for pregabalin in the regulation of chaperone activity and cytoskeleton remodelling in glial cells.
Although the significant up-regulation of calreticulin and DJ-1 protein was confirmed in Western blot analysis, its extent was not compatible with that of the two-dimensional gel data. In the latter, these proteins showed greater differences than in the Western blot analysis, which will require further study. However, we have in the meantime ascribed the differences to a post-translational thiol modification of the proteins.
Calreticulin is a chaperone that binds to oligosaccharides on incompletely folded proteins and retains them in the endoplasmic reticulum (Spiro et al., 1996). Like other chaperones, calreticulin prevents incompletely folded proteins from undergoing irreversible aggregation. However, the biological significance of calreticulin is controversial. For example, it has been demonstrated that overexpression of calreticulin enhances sensitivity to cell death (Nakamura et al., 2000; Arnaudeau et al., 2002). In contrast, calreticulin is up-regulated in response to oxidative stress; overexpression of calreticulin prevents cell death mediated by oxidative stress (Liu et al., 1998; Nunez et al., 2001). Up-regulation of calreticulin in 6-OHDA (6-hydroxydopamine)-treated cells may also comprise an active decision by neurons to impart cellular tolerance to stresses and hinder the subsequent propagation of cell death signals (Lee et al., 2003). DJ-1 was identified as a novel oncogene and later found to be a causative gene for a familial form of Parkinson's disease, PARK7 (Nagakubo et al., 1997; Bonifati et al., 2003). DJ-1 is a multi-functional protein that participates in anti-oxidative stress reactions, transcriptional regulation and chaperone reaction (Yokota et al., 2003; Canet-Aviles et al., 2004; Martinat et al., 2004; Shendelman et al., 2004; Taira et al., 2004; Zhong et al., 2006).
Most neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease and other polyglutamine diseases, are associated with degeneration and death of specific neuronal populations due to misfolding or aggregation of certain proteins (Gao et al., 2008). These aggregates often contain ubiquitin that is the signal for proteolysis by the ubiquitin–proteasome system, and chaperone proteins that are involved in the assistance of protein folding (Gao et al., 2008). We do not yet know the elaborate mechanisms of chronic pain syndrome. The specific role of glia proliferation in pain control has not been clearly demonstrated. Nevertheless, increasing numbers of glial cells, such as microglia, may result in increasing production of inflammatory mediators, leading to abnormal pain (Suter et al., 2007).
Pregabalin and gabapentin reduce nociceptive behaviour in animal models of neuropathic pain or inflammation (Xiao et al., 1996; Field et al., 1997; Houghton et al., 1998; Partridge et al., 1998; Taylor et al., 1998; Chen et al., 2001; Takasaki et al., 2001). Pregabalin and gabapentin alter the neuropathic pain state, suggesting that their antinociceptive action is dependent on alterations that occur specifically in neuropathic or inflammatory conditions. Pregabalin affected chaperone and cytoskeleton proteins, suggesting the possibility that chronic pain is associated with changes in the cytoskeleton array and protein folding, and also that the adverse effect of this change is the mechanism by which pregabalin operates. Especially, calreticulin and DJ-1 were consistently regulated by both pregabalin and gabapentin. Also, pregabalin weakened the formation of web-like F-actin filaments. In agreement with the results of the present study, gene ontology analysis showed that allodynic-regulated genes are involved in a variety of processes, including myelination, actin cytoskeleton reorganization, dephosphorylation and phosphorylation responses to stress, as well as protein trafficking and RNA processing (Coyle et al., 2007). In addition, the mechanisms of a small family of GDP/GTP exchange factors, Epac (exchange protein directly activated by cAMP), acting on small G-proteins is involved in the regulation of cytoskeletal elements (Hucho et al., 2005). While a direct link between Epac and the cytoskeleton in nociceptor signalling has not been established, long-time drug treatment inducing stabilization, as well as destabilization of microtubules, produces painful neuropathies in patients and neuropathic pain-like behaviour in animals (Aley et al., 1996; Dina et al., 2001). In contrast, short-term destruction of cytoskeletal components dramatically attenuates the establishment of mechanical hyperalgesia (Dina et al., 2003).
Joomin Lee and Seyeon Park performed the two-dimensional experiments and protein analysis. Seyeon Park designed the experiments, analysed the data and wrote the manuscript.
This work was supported by the
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Received 8 May 2009/11 June 2009; accepted 7 September 2009
Published as Cell Biology International Immediate Publication 7 September 2009, doi:10.1042/CBI20090018
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 C6 cell protein patterns after treatment with pregabalin as measured by two-dimensional gel electrophoresis