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1 Department of Neurology, and 2 Department of Molecular Cell Biology, Institute of Advanced Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan
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Abstract |
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Introduction |
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37 kDa molecule that exists as a tetramer in the cell (Sirover 1999; Berry & Boulton 2000). It localizes in multiple subcellular compartments, including the plasma membrane, nucleus and cytoplasm (Schmitz 2001). GAPDH is well known to play an important role as a glycolytic enzyme, converting glyceraldehydes-3-phosphate into 13-bisphosphoglycerate. But it has recently been shown to have various functions, such as nuclear RNA export, DNA replication and DNA repair in addition to the classical glycolytic activity (Sirover 2005). Pro-apoptotic activity is another biological function in which GAPDH is proposed to play a part, but this has not proven to be causative (Chuang et al. 2005; Kim & Dang 2005; Sirover 2005). In most of the cells studied, GAPDH is originally present in the cytoplasm, but appears to accumulate in the nucleus from the early phase of apoptosis (Ishitani et al. 1997; Sawa et al. 1997; Chuang et al. 2005). One group reported that immuno-detectable GAPDH appearance in the nucleus appears to precede the occurrence of apoptotic nuclear damage (Carlile et al. 2000). However, another study using cells that express GAPDH either little or half in the nucleus suggests that the presence of GAPDH in the nucleus does not appear to cause apoptosis (Dastoor & Dreyer 2001). They propose that GAPDH might have an activity of a nuclear carrier for other pro-apoptotic molecules. Furthermore, over-expression of Bcl-2, which interacts directly with the mitochondrial voltage-dependent anion channel and inhibits the release of death signal from mitochondria, blocks both apoptosis and nuclear GAPDH translocation (Maruyama et al. 2001). This finding suggests that the nuclear GAPDH localization is a later event of apoptosis downstream of the apoptosome formation and caspase activation.
GAPDH is also apparently implicated in the pathogenesis of neurodegenerative diseases. It binds to the polyglutamine repeats of huntingtin that cause Huntington's disease (Burke et al. 1996). GAPDH is found in the amyloid plaques of Alzheimer's disease (Sunaga et al. 1995), and associated with apoptosis in the postmortem nigral neuronal nuclei of the patients with Parkinson's disease (Tatton et al. 2003; Tsuchiya et al. 2005). However, association or presence of a molecule with the pathognomonic structure related with a specific disease does not necessarily implicate the function connected with the pathogenesis of a disease. Therefore, the function of GAPDH in the pathogenesis of these neurodegenerative diseases is not clear yet.
In the current study, we have investigated the effect of 1-Methyl-4-phenyl-pyridium iodide (MPP+), an inhibitor of the complex I of the mitochondrial electron transfer system, on the translocation of GAPDH and on apoptosis. We have also studied whether localization of GAPDH in the nucleus by expressing it as a fusion molecule with nuclear localization signal (NLS) might influence the occurrence of apoptosis. The recent finding of the CRM1-mediated nuclear export signal in the C-terminal domain of GAPDH (Brown et al. 2004) allowed us to use a specific inhibitor of this export pathway for dynamically assessing the function of GAPDH nuclear localization.
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Results |
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To detect the movement of GAPDH from the cytoplasm to the nucleus, we stably expressed GAPDH C-terminally fused with EGFP (GAPDH-EGFP) in SH-SY5Y cells using an eukaryotic expression vector, and exposed them to 5 mM MPP+ for various periods. As shown in Fig. 1B, cells before incubation with MPP+ showed almost exclusive localization of GAPDH-EGFP in the cytoplasm, and after 6 h of incubation with MPP+, they showed a sign of nuclear transfer of GAPDH-EGFP from the lower part of the nucleus. The nuclear membrane appeared to be almost intact, but there was minute deformation at the lower part of the nucleus. After 12 h and 24 h of incubation, cells exhibited typical signs of apoptosis and clear nuclear accumulation of GAPDH-EGFP.
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Several groups have reported that the detection of GAPDH-EGFP in the nucleus precedes the occurrence of apoptosis (Chuang et al. 2005). However, the above finding might suggest an opposite order of events. Apoptotic nuclear membrane damage appears to precede the occurrence of GAPDH entry into the nucleus. Therefore, we examined in the current study whether the enzyme has an apoptotic function in neuronal cells by over-expressing GAPDH with or without NLS using a retrovirus vector. The exogenously expressed GAPDH was tagged with a Flag epitope, so that the localization can be detected easily using an immunostaining with anti-Flag antibody as the first antibody followed by a confocal imaging. We used two neuronal cells with distinct expression patterns of endogenous GAPDH. Endogenous GAPDH of SH-SY5Y cells is known to localize in the cytoplasm under normal conditions (Maruyama et al. 2002), whereas the enzyme distributes equally in the nucleus and in the cytoplasm in NB41A3 cells (Dastoor & Dreyer 2001).
As shown in Fig. 2, exogenously expressed GAPDH with NLS at its C-terminus (GAPDH-NLS) was highly concentrated in the nucleus of SH-SY5Y cells (Fig. 2A), whereas the same molecule without NLS localized in the cytoplasm diffusely (Fig. 2B). These findings suggest that NLS is functional, and that GAPDH can be expressed in the nucleus of the cells that normally express it exclusively in the cytoplasm. GAPDH-NLS showed a little different expression pattern in NB41A3 cells; the molecule distributes strongly at the peripheral region in the nucleus with diffuse expression in the cytoplasm (Fig. 2C). However, GAPDH without NLS was exclusively expressed in the cytoplasm as in SH-SY5Y cells (Fig. 2D). Along the course of establishing these cells expressing GAPDH using a retrovirus vector, we did not recognize any apoptosis-specific morphological change or retardation of the proliferative activity.
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GAPDH is reported to participate in apoptosis of neuronal cells. Since we could not observe induction of apoptosis by exogenous expression of GAPDH in the nucleus of two neuronal cells, we speculated that a trigger to induce apoptosis might potentiate the apoptotic function of GAPDH. 1-Methyl-4-phenyl-pyridium iodide (MPP+) is an inhibitor of the complex I of the mitochondrial electron transfer system and is known to induce neuronal apoptosis by causing cytochrome c release from mitochondria. To titrate the dose of the reagent and the incubation time for the cell survival, we used 0.19–45 mM MPP+ for SH-SY5Y and NB41A3 cells expressing EGFP (vector alone), GAPDH-Flag, and GAPDH-NLS-Flag, and incubated them for 24 and 48 h. As shown in Fig. 3, both SH-SY5Y and NB41A3 cells did not show any statistically significant survival difference among three different transductants at any MPP+ concentrations in either incubation period.
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While incubating with MPP+, we found rounded cells detaching from the plates with more such cells at higher concentrations of MPP+ and after longer incubation periods. To verify the mechanism of cell death, the rounded detached cells were collected and stained with Annexin-V and Hechst33258 (Fig. 4). Most of the cells were positive for Annexin-V staining, and showed fragmented nuclei in Hechst33258 staining, which are typical of apoptotic cells.
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The presence of molecules inside and outside of the nucleus is controlled by the molecular systems mediating nuclear import and export. Recently, one of the export systems has been found to be operative in determining the GAPDH localization. They found the presence of CRM1-dependent nuclear export signal comprised of 13 amino acids in the C-terminal domain of GAPDH (Brown et al. 2004). The CRM1-dependent nuclear export can be inhibited by leptomycin B (LMB). Therefore, we examined whether neuronal cells exogenously expressing GAPDH can be induced to apoptosis by leptomycin B treatment (Fig. 6A). In the presence of LMB (5 µM), SH-SY5Y cells with or without exogenous expression of GAPDH did not show any difference in apoptotic pattern and extent including the ladder formation at both 6 h (lanes 2–4) and 24 h (lanes 5–7) of incubation. The presence of NLS fused to GAPDH (lanes 4 and 7) did not affect apoptosis at all. The DNA ladder formation became obvious after 24 h of incubation with LMB alone, suggesting an apoptotic effect of LMB itself.
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Function of exogenously expressed GAPDH
Since GAPDH-Flag and GAPDH-NLS-Flag are exogenously expressed proteins, their enzymatic characteristics might be different from the intrinsic GAPDH. The enzymatic function of exogenous enzymes might be responsible for explaining the apparent absence of difference in induction of apoptosis. We have analyzed the glycolytic activity by studying Michaelis-Menten kinetics. As shown in Fig. 7A, SH-SY5Y cells transduced with GAPDH-Flag and GAPDH-NLS-Flag had higher initial reaction velocity per same amount of total soluble cellular protein over control cells, suggesting the presence of exogenous GAPDH. To examine the functional characteristics of the exogenous protein, we have re-plotted the values for Lineweaver-Burke Plot and obtained the Km values for three different transductants. The three yielded similar values of –1/Km = 0.20–0.22 mM–1, so that the Km values were calculated to be 4.5–5.0 mM under the conditions we used. These findings suggest that the exogenously expressed GAPDH-Flag and GAPDH-NLS-Flag are as active as the intrinsic GAPDH.
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Discussion |
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A previous study shows that, under an apoptotic stress, the immunoreactive GAPDH appears inside the nucleus before the start of morphologically detectable apoptosis (Carlile et al. 2000). However, the immunocytochemical techniques are not sensitive enough to detect the condition of nuclear pores that regulate the pass of GAPDH into the nucleus. Therefore, the time-course observation detected using these techniques does not necessarily reflect the molecular derangements occurring early in the apoptotic process. We could not detect translocation of GAPDH into the nucleus before the onset of apoptosis. Therefore, we have attempted in the current study to resolve these technical problems by exogenously expressing the molecule with or without NLS fused to the C-terminus of GAPDH. NLS changed the cellular distribution of GAPDH with more NLS-fused molecules in the nucleus. However, the nuclear expression of GAPDH itself did not cause apoptosis. Exposure of the cells exogenously expressing GAPDH to MPP+ caused apoptosis, but no quantitative difference was detectable among the different transductants expressing vector alone, GAPDH without NLS and GAPDH with NLS at any time points examined. These findings suggest that the presence of GAPDH itself in the nucleus is not responsible for making cells prone to apoptosis. The apparent appearance of the molecule before clear nuclear deformation might be taken as a result but not a cause of minute structural damages to the nuclear membrane that were not easily detectable by immunocytochemistry.
From our studies, two things have become clear: firstly, MPP+ is not responsible for translocation of GAPDH into the nucleus before the onset of apoptosis and secondly, a simple translocation of GAPDH into the nucleus is not responsible for apoptosis. One possibility for explaining the discrepancy between the previous reports and ours might be that the dynamic transfer of GAPDH from the cytoplasm to the nucleus is more important than the localization itself as suggested by a study (Dastoor & Dreyer 2001). GAPDH might carry other proteins into the nucleus which cause apoptosis. Recent finding that GAPDH has a CRM1-dependent nuclear export signal in the C-terminal domain (Brown et al. 2004) provided a way of testing the function of GAPDH dynamically using a specific inhibitor of the export system, LMB. We have hypothesized that exposure of the cells exogenously expressing GAPDH to LMB induces more extensive transfer of GAPDH into the nucleus leading to stronger apoptosis. Our results, however, indicated that LMB treatment did not change the extent of apoptosis at all. Even additional treatment with MPP+ did not change the time course or extent of apoptosis. In addition, we observed that LMB itself caused apoptosis after a 24-h incubation. These findings suggest that MPP+ is not an inducer of nuclear translocation of GAPDH, and that some molecules responsible for apoptosis might be trapped in the nucleus after LMB treatment. We further speculate that nuclear translocation of GAPDH is not involved in the initiation of MPP+-induced apoptosis.
We conclude that the shift of GAPDH from the cytoplasm to the nucleus induced by NLS fusion does not cause apoptosis, and that the nuclear translocation of GAPDH in response to a respiratory insult, MPP+, is highly likely to be the result of early minute cellular damage caused by apoptosis.
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Experimental procedures |
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SH-SY5Y cells derived from human dopaminergic neuroblastoma were grown in Cosmedium-001 culture medium (CosmoBio, Tokyo, Japan) supplemented with 5% fetal bovine serum (Equintech-Bio, TX, USA). NB41A3 cells (ATCC, Bethesda, MD, USA) derived from mouse neuroblastoma were grown in F12K culture medium containing 15% horse serum (Equintech-Bio) and 2.5% fetal bovine serum (Equintech-Bio).
Establishment of cells over-expressing recombinant GAPDH
For construction of an expression vector for GAPDH C-terminally fused with enhanced green fluorescence protein (GAPDH-EGFP), the cDNA for human GAPDH without termination codon was obtained from the total RNA of HEK293 cells with a pair of primers, 5'-CGGGATCCAGTCAGCCGCATCTTCTTTTG-3' (forward, containing BamHI site at the 5' end) and 5'- CCCAAGCTTCTCCTTGGAGGCCATGTGGGC-3' (reverse, containing HindIII site at the 5' end). The product was digested with BamHI and HindIII, and inserted in frame with EGFP into a plasmid, pcDNA3.1, that carries the expression cassette of EGFP with a HindIII site at the 5' end and a hygromycin-resistant gene. Following the FuGENE 6 (Roche, Penzberg, Germany) -mediated transfection of the vector, stably transfected SH-SY5Y cells were selected using hygromycin.
For construction of GAPDH-NLS-Flag in a retrovirus vector, the cDNA encoding human GAPDH without the termination codon was obtained from the HEK293-cell total RNA employing RT-PCR with a pair of primers, 5'-CGGGATCCAGTCAGCCGCATCTTCTTTTG-3' (forward, containing BamHI site at the 5' end) and 5'-CGGCTCGAGGCTAGCCTCCTTGGAGGCCATGTGGGC-3' (reverse, containing XhoI and NheI sites at the 5' end). The DNA encoding SV40 nuclear localization signal (NLS) was obtained from the pECFP-Nuc vector (Clontech, Palo Alto, CA, USA) using PCR with a pair of primers, 5'-CGCTCGAGGCTAGCGTCAGATGTCGAGCTGATCCAAAAAAG-3' (forward, containing XhoI and NheI sites at the 5' end) and 5'-GTCTAGAAAGCTTCGATCCTACCTTTCTCTTC-3' (reverse, containing XbaI and HindIII sites at the 5' end). These PCR products were subcloned into a pCR BluntII TOPO vector (Invitrogen, San Diego, CA, USA), and named TOPO/GAPDH and TOPO/NLS. The DNA coding for GAPDH was excised out from TOPO/GAPDH by digesting with BamHI and NheI, and the DNA fragment was ligated in frame with the NLS-coding DNA in TOPO/NLS that was digested with BamHI and NheI. We named this construct TOPO/GAPDH-NLS. The insert, GAPDH-NLS, that was obtained after digesting TOPO/GAPDH-NLS with BamHI and HindIII, was subcloned into a eukaryotic expression vector, pcDNA3.1/Flag, in frame with the Flag-encoding sequence. pcDNA3.1/Flag contains a DNA fragment encoding a Flag epitope preinserted in the region of multiple cloning sites with HindIII at its 5' end. Then, pcDNA/GAPDH-NLS-Flag was again digested with BamHI and XhoI to yield an insert, GAPDH-NLS-Flag, and the insert was cloned into a vector, pMXs-IG, which in advance was digested with BamHI and XhoI.
To construct a retrovirus vector expressing GAPDH-NLS-Flag and enhanced green fluorescent protein (EGFP) from the same transcript that is transcribed from the DNA containing an internal ribosomal entry site (IRES) between the two cDNAs, pMXs/GAPDH-NLS-Flag-IG and pCAG-VSVG were co-transfected into a packaging cell line, HEK293gpIRES, using the FuGENE 6 lipofection method (Kitamura et al. 2003). The retrovirus created in HEK293gpIRES cells was concentrated, and used for infection of SH-SY5Y and NB41A3 cells at M. O. I. of 8. We established SH-SY5Y and NB41A3 cells stably over-expressing the GAPDH-NLS-Flag fusion protein. The retrovirus vector that expresses EGFP and the GAPDH-Flag fusion protein without NLS, and the one that expresses only EGFP were also generated using similar methods, and the cells stably expressing these molecules were then created.
Immunostaining
For immunostaining of GAPDH-Flag, cells were seeded at 5 x 104 cells/well into a 12-well plate coated with 0.01% poly L-lysine. Two days later, cells were washed twice in PBS and fixed in 3.7% paraformaldehyde, pH 7.4, for 10 min. They were then washed 3 times in PBS, and permeabilized with 0.2% Triton X-100/PBS for 5 min on ice. Then cells were washed 3 times in PBS containing 1% normal goat serum, and the first antibody, anti-Flag (Sigma, St. Louis, USA) was added, and incubated at room temperature for 1 h. Cells were washed 3 times in PBS containing 1% normal goat serum, and cells were incubated with the second antibody conjugated with AlexaFluor 568 (Invitrogen), at room temperature for 1 h, and mounted with GEL/MOUNT (Biomeda, Foster City, CA, USA).
For detecting apoptotic cells, we used AlexaFluor568-conjugated Annexin-V (Annexin-V-Alexa 568) that binds to phosphatidylserine, and Hoechst 33258 for detecting morphological changes of nuclei of dead cells. Cells were seeded at 106 cells/well into a 6-cm plate. They were then incubated for 24 h with 5 mM of MPP+ at 37 °C, in 5% CO2 atmosphere, collected, and washed with PBS. After centrifugation at 200 g for 5 min, the cell pellet was resuspended and incubated for 10–15 min at room temperature in 100 µL of an incubation buffer, which contains 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl and 5 mM CaCl2. Annexin-V-Alexa 568 (Roche, Penzberg, Germany), 20 µL, was prediluted in 1 mL of the incubation buffer before adding into the cell suspension. For staining the nuclei, cells were collected and washed with PBS, and incubated for 10 min with 100 µL of PBS containing 0.25 mM Hoechst 33258 (DOJINDO, Kumamoto, Japan). The stained cells were mounted on a slide glass with GEL/MOUNT. Immunofluorescence was detected using a Nikon fluorescence microscope Eclipse E800, a Nikon inverted microscope Eclipse TE300 or a confocal microscopy system equipped with Bio-Rad Radiance 2000.
Cell viability assay
Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, San Luis Obispo, CA, USA). Briefly, SH-SY5Y or NB41A3 cells were seeded at 5000 cells/well into 96-well plates (Corning, Acton, MA, USA). Next day, an apoptotic toxin, 1-Metyl-4-phenyl-pyridium iodide (MPP+) (Sigma) in water was added to each well to make final concentrations of 0.185 mM, 0.556 mM, 1.667 mM, 5 mM, 15 mM, and 45 mM after 100-fold dilution in a final volume of 100 µL. The MTS assay solution (20 µL) was then added into each well, 24–48 h later. After 2-h incubation with the solution at 37 °C, 5% CO2 atmosphere, the absorbance was measured at 490 nm using a 96-well plate reader (MTP-32) (CORONA, Tokyo, Japan).
Detection of DNA fragmentation
To detect the DNA ladder generated by apoptosis, 1.5 x 106 cells were collected by scraping and centrifugation, and lyzed by incubating them with a 600-µL solution containing 1% SDS, 0.5 mg/mL proteinase K, and 0.5 mg/mL RNaseA at 37 °C for 30 min. The lysate was then mixed with 900 µL of NaI solution containing 6 M NaI, 13 mM EDTA, 0.5% SDS, 10 mg/mL glycogen and 26 mM Tris-HCl (pH 8.0), and incubated at 60 °C for 15 min. Then, isopropanol, 1.5 mL, was added, and the mixture was incubated at room temperature for 15 min. After centrifugation at 20 000 g for 15 min, DNA pellet was rinsed twice with 50% isopropanol and once with 100% isopropanol. The pellet was washed with diethylether, air-dried, and dissolved in 50 µL solution containing 10 mM Tris-HCl and 1 mM EDTA (pH 7.5). The DNA sample, 500 ng, was fractionated using 2% agarose gel in TBE buffer. DNA was visualized by staining with ethidium bromide.
GAPDH assay
GAPDH assays were performed according to the method described (Beutler 1971; Koren et al. 2001). SH-SY5Y cells expressing GAPDH-Flag, GAPDH-NLS-Flag and EGFP (control) were harvested by scraping from culture plates in PBS containing 5 mM EDTA after washing with PBS. After centrifugation, cells were resuspended in PBS containing 0.25 M sucrose, frozen at –80 °C, and lyzed by sonication on ice. The supernatant was collected, aliquoted in small amounts, and frozen at –80 °C. Total protein concentration was measured using a BCA protein assay kit from Pierce in each supernatant, and the supernatant with the same protein amount (10 µg) was used for enzyme assay.
The assay buffer contained the following: 1 mM NAD, 15 mM disodium arsenate (pH 8.0), 1.35 mM EDTA and varying concentrations of D-glyceraldehyde-3-phosphate (substrate) in 100 mM Tris-HCl (pH 8.0). The enzyme activity was determined by incubating the aliquot of cell supernatant in 1 mL of the assay buffer at 37 °C followed by measurement of the optical density at 340 nm (OD340). The assays were performed in quintuplicate, and the mean values were used for analysis. The initial time-course study of the enzyme activity gave a linear activity increase up to 5 min. Therefore, the increase of OD340 (
OD340) in 1 min was taken as the initial reaction velocity (V0).
Statistical analysis
Statistical analysis was carried out using MANOVA of the JMP software (SAS Institute Inc., Cary, NC, USA).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: t-kondo{at}wakayama-med.ac.jp; ksaka{at}wakayama-med.ac.jp
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References |
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Received: 16 June 2005
Accepted: 26 September 2005
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