1 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 180-8611, Japan
*Author for correspondence (e-mail: ikegaya{at}tk.airnet.ne.jp)
Accepted July 9, 2001
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SUMMARY |
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Key words: Astrocyte, GLT-1, Basic fibroblast growth factor, Epidermal growth factor, Insulin, Platelet-derived growth factor, Hepatocyte growth factor
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INTRODUCTION |
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Recent observations indicated different developmental patterns of GLT-1 and GLAST expression (Shibata et al., 1996; Sutherland et al., 1996; Bar-Peled et al., 1997; Furuta et al., 1997b; Ullensvang et al., 1997). GLAST expression diminishes following the completion of cell migration during embryogenesis, whereas GLT-1 increases progressively to adult levels during postnatal maturation. Pathophysiological study shows that GLT-1 mRNA levels are lowered in postischemic rat hippocampus, suggesting a possible mechanism for the decreased clearance of glutamate in ischemia models (Torp et al., 1995). Despite such dynamic changes in the expression of the glutamate transporters, little is known about mechanisms underlying these patterns of expression. When immature astrocytes bearing only GLAST are cocultured with neurons or treated with cAMP analogs, they express GLT-1 with the augmented expression of GLAST (Swanson et al., 1997; Schlag et al., 1998). Fimbria-fornix or corticostriatal lesion induces a decrease in immunoreactivity for GLAST as well as GLT-1 within the hippocampus and striatum (Ginsberg et al., 1995). These results suggest that neuronal factors are involved in the induction of GLT-1 and GLAST expression. However, very few endogenous molecules have so far been identified as intercellular regulators of the glutamate transporter.
Of extracellular soluble factors derived from neurons, growth factors are well known to modulate the morphology and functions of astroglial cells. Zelenaia et al. recently showed that epidermal growth factor (EGF) induces GLT-1 expression in immature astrocytes (Zelenaia et al., 2000). Therefore, we have focused the present study on the effects of growth factors on GLAST expression. Using primary astrocyte cultures prepared from rat cerebral cortex, we demonstrate that, of the six growth factors tested (basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), EGF, insulin, platelet-derived growth factor AB (PDGF-AB), and hepatocyte growth factor (HGF)), bFGF, IGF-1 and EGF enhance the transcriptional level of GLAST in a concentration- and time-dependent manner.
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MATERIALS AND METHODS |
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Glutamate uptake
The culture media were replaced with a modified Hanks balanced salt solution (HBSS) containing 2 mM glucose (pH 7.2). After a 30 minute preincubation, each culture received treatment with 0.025 µCi/ml L-[3H]-glutamate (Amersham Pharmacia Biotech, Uppsala, Sweden) and unlabeled glutamate to achieve final glutamate concentrations in the range of 2 µM to 200 µM. Uptake was terminated after 7-minute incubation at 37°C by three washes in ice-cold HBSS, immediately followed by cell lysis in 1 N NaOH. Aliquots were taken for scintillation counting and for Lowrys protein assay (Lowry et al., 1951) using BSA standards. Blanks prepared from osmotically lysed cells revealed that glutamate binding showed <0.3% of the 3H accumulation.
Immunoblot analysis
Affinity-purified polyclonal antibodies to GLAST and GLT-1 were generous gifts from K. Tanaka (Tokyo Medical and Dental University). The specificity of these antibodies was previously reported (Shibata et al., 1997; Yamada et al., 1998).
Cultures were washed twice with cold phosphate-buffered saline (PBS), plates were scraped, and cells were suspended in 0.5 mM EDTA-PBS. The suspensions were centrifuged at 10,500 g at 4°C for 5 minutes. The pellet was resuspended in buffer containing protease inhibitors (25 µg/ml leupeptin, 25 µg/ml pepstatin, 1.25 mM phenylmethylsulfonyl fluoride and 0.5 mM EDTA) and homogenated on ice. The suspensions were then centrifuged at 300 g at 4°C for 10 minutes, and protein solutions were obtained. Proteins were denatured by heating at 70°C in 5% 2-mercaptoethanol for 20 minutes. Stacking gels (5% acrylamide) were loaded with 3 µg protein and molecular weight markers, and run at 50 V for 1 hour, then run through a 11% acrylamide gel at 100 V for 2 hours. Proteins were electrophoretically transferred to a polyvinylidenefluoride membrane (Millipore, Bedford, MA) at 80 mA for 15 hours. The membranes were stored in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat milk for 1 hour. After three washes, primary antibodies (anti-GLAST antibody 1 µg/ml, anti-GLT-1 antibody 0.5 µg/ml) were applied in Tris-buffered saline/0.1% Tween-20 (TBS-T) containing 5% nonfat milk for 1 hour at room temperature. Excess primary antibody was removed with three additional washes, and the membranes were incubated with an alkali phosphatase-conjugated anti-rabbit secondary antibody in TBS-T containing 5% nonfat milk at room temperature for 1 hour. Antibody binding was detected by an ECF western blotting detection kit (Amersham).
Immunohistochemistry
Cultures were washed twice in PBS, and fixed with 4% paraformaldehyde at room temperature for 10 minutes. After two rinses, cells were treated with 0.1% Triton X-100 at room temperature for 30 minutes. Cultures were incubated with rabbit polyclonal antibodies for GLAST (1:1000) at 4°C overnight. After being washed in PBS, coverslips were incubated in PBS containing 10 µg/ml propidium iodide and anti-rabbit IgG-FITC conjugates (1:1000) at room temperature for 4 hours. Confocal imaging was carried out with a laser scanning confocal system Micro Radiance (Biorad, Herculeus, CA) equipped with an inverted microscope ECLIPSE TE300 (Nikon, Tokyo, Japan), an argon ion laser and a host computer system. All imaging and processing operations were performed with Laser Sharp Acquisition (Biorad) and Laser Sharp Processing (Biorad), respectively. To observe propidium iodide and FITC fluorescence, the cultures were illuminated with an excitation wavelengths of 488 and 514 nm, and the fluorescence images were obtained through a 500- and 570-nm band-pass filter, respectively.
Preparation of cDNA probe
Antisense oligonucleotides for GLAST and GLT-1were prepared for northern blots. Reverse transcription of mRNA to generate cDNA was accomplished using reverse transcriptase and random hexamer (Superscript Amplification System, Life Technology, Grand Island, NY). A total of 2-4 µl of rat brain cDNA was used for PCR amplification in the presence of 2.5 U Taq DNA polymerase (Takara, Otsu, Japan) and specific primers. Sequences of the primers were 5'-CTCACTGACTGTGTTTGGTG-3' and 5'-GAGGTGCCACCAGAACTTTC-3' (457 bp product size) for GLT-1 and 5'-ATGCCTTTGTGCTACTCACC-3' and 5'-GTGTTTCGTTGGCCTGGATG-3' (460 bp) for GLAST. Thermal cycling was performed as follows: one cycle at 94°C (30 seconds), then 35 cycles at 94°C (10 sec), 55°C (30 seconds), and 72°C (30 seconds), followed by 72°C (5 minutes) for final extension. Amplification without templates was used as a negative control. The PCR products were separated by electrophoresis on 1% agarose gels, and then stained with ethidium bromide. The bands were photographed under UV light. The PCR-amplified products were subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and used for transformation of INVF competent cells. Positive transformations containing plasmids with inserts were selected by growing bacteria on LB agar plates containing 50 µg/ml ampicillin and 2% X-gal. Plasmid DNA was isolated from minicultures and digested with EcoRI. Plasmids containing the PCR products were then sequenced.
Northern blot analysis
Astrocytes of 14 flasks (75 cm2) were pooled to obtain about 2 µg mRNA for each group. mRNAs were isolated from cultured astrocytes by oligo-dT selection using the mRNA purification kit (Qiagen, Valencia, CA). The mRNA samples and RNA size markers (BRL, Grand Island, NY) were electrophoresed on 2.2 M formaldehyde denaturing 1% agarose gel and blotted onto a nitrocellulose membrane. The membrane was crosslinked in an UVStratalinker 1800 (Stratagene, La Jolla, CA). Antisense oligonucleotides prepared as described above were labeled with [32P] and hybridized to each membrane at 42°C for 15 hours in buffers consisting of 50% deionized formamide, 1% sodium dodecyl sulfate, 4x standard saline phosphate EDTA, 2x Denhardts solution, and 10 µg salmon sperm DNA. After hybridization, the membranes were washed with increasing stringency at 37-55°C, and autoradiographs were developed with Kodak Biomax MS film (Eastman Kodak, Rochester, NY). After stripping, the membranes were hybridized to a labeled GAPDH probe to produce second autoradiograph.
Statistical analyses
Statistical analyses were performed with Students t-test for comparisons between two groups or Tukeys test following one-way ANOVA for multiple comparisons. All results are expressed as means±s.e.m.
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RESULTS |
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Similar facilitatory effects were observed when either IGF-1 or EGF was applied for 24 hours (Fig. 2). However, no apparent effect was obtained for treatment with insulin, PDGF-AB or HGF. Taken together, these results suggest that some, but not all, growth factors promote the glutamate transporter activity in cultured cortical astrocytes.
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Transient GLAST induction following treatment with growth factors
We assessed the time course of the facilitatory effects of growth factors. When bFGF was continuously applied to astrocytes, the increase in glutamate uptake activity was observed after 24-42 hours. The effect reached a peak after 36 hours, and rapidly returned to baseline by 48 hours (Fig. 6A). The effect of IGF-1 and EGF also returned to basal levels after 48 hours (mean percent of baseline: 106.9±10.4% and 93.5±4.5% at 48 hours, respectively, n=4). These results suggest that these growth factors exert their effects within a narrow time window.
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Growth factors activate different signaling pathways for GLAST upregulation
To determine whether bFGF, IGF-1 and EGF share a common signaling pathway for the GLAST induction, we investigated the effect of various combinations of the growth factors on glutamate uptake activity (Fig. 7). In this series of experiments, bFGF, IGF-1 and EGF were applied at concentrations of 10 ng/ml, 100 ng/ml and 30 ng/ml, respectively, because these high concentrations rendered the maximal effect on the glutamate uptake (data not shown). A combination of bFGF and IGF-1 caused further increase in the maximal activity of glutamate uptake, compared with treatment with bFGF or IGF-1 alone. Likewise, a combination of IGF-1 and EGF induced an additive increase in the glutamate uptake. However, a combination of bFGF and EGF did not show such an additive effect. The effect of a mixture of all three growth factors was similar to that of a combination of IGF-1 with either bFGF or EGF, whereas it was significantly larger than that of a combination of bFGF and EGF. The data suggest that the effects of these growth factors are mediated, at least in part, by different signaling mechanisms. Therefore, we finally performed pharmacological investigations on the growth factor-mediated GLAST induction (Table 1).
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Growth factors are also known to stimulate the Ras/mitogen-activated protein kinase (MAPK) pathway and the phosphatidyl inositol 3-kinase (PI3K) pathway (Leof, 2000). Indeed, N-acetyl-S-farnesyl-L-cysteine (AFC), an inhibitor of Ras, significantly abrogated the effects of bFGF, IGF-1 and EGF. Similarly, either the inhibitor of MAPK kinase PD98059 or the inhibitor of PI3K wortmannin blocked the effects of all three growth factors. Therefore, bFGF, IGF-1 and EGF may activate both Ras/MAPK and PI3K pathways in order to modulate GLAST expression.
Although S6 kinase (Volarevic and Thomas, 2000) and phospholipase C (PLC) (Kamat and Carpenter, 1997) have also been identified as downstream signaling molecules of growth factor receptors, neither the inhibitor of S6 kinase rapamycin nor the inhibitor of PLC U73122 blocked the effect of the growth factors.
Intracellular Ca2+ is known as a second messenger of gene regulation and thereby induce diverse transcriptional responses (Bading et al., 1997). The possible contribution of Ca2+ to the GLAST regulation was assessed by examining the effect of the intracellular Ca2+ chelator BAPTA-AM. BAPTA-AM abolished the effect of bFGF, but the effect of IGF-1 or EGF were substantially unaffected. The [Ca2+]i elevation may be required only for the bFGF receptor signaling. Therefore, we investigated the involvement of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), both of which are involved in Ca2+-activated signaling pathway. Unexpectedly, the inhibitor of PKC calphostin C had no influence on the effect of bFGF but rather blocked the effect of IGF-1 and EGF. The inhibitor of CaMKII KN93 had no effect on growth factor-increased glutamate uptake. Incidentally, none of these inhibitors used in this study affected the baseline glutamate uptake activity.
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DISCUSSION |
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In the primary cultures, astrocytes show a polygonal morphology and express only GLAST. Expression of the other astrocytic transporter GLT-1 is generally thought to depend on neuron-derived factors (Swanson et al., 1997). Indeed, Zelenaia et al. reported that long-term treatment with EGF, an assumed neuronal signal, can induce the GLT-1 expression in immature astrocytes (Zelenaia et al., 2000). However, the neuronal signal is not unique to the GLT-1 regulation. The observation that destruction of glutamatergic corticostriatal projections causes the downregulation of GLAST as well as GLT-1 (Levy et al., 1995) suggests that normal astroglial/neuron interactions are also important for the GLAST regulation. However, molecular mechanisms underlying the GLAST regulation have not been elucidated. In the present study, bFGF, IGF-1 and EGF induced an increase in GLAST expression levels. GLT-1 expression was unchanged by these growth factors for at least 0.5 to 48 hours of exposure.
Despite the continuous presence of the growth factors, the GLAST upregulation was a transient response. Growth factor-mediated increases in glutamate uptake activity were well accompanied by changes in GLAST mRNA levels, which suggests that the growth factors modulate the transcriptional level. Indeed, the slightly preceding increase in GLAST mRNA may reflect a translational process. Another possibility is that the increased GLAST mRNA is due to reduced mRNA degradation. In any case, however, the transient regulation by growth factors may be a key property that may allow a dynamic and delicate control of GLAST expression in astrocytes. In contrast with the previous report (Zelenaia et al., 2000), we found no evidence that EGF induced GLT-1 expression. This inconsistency may be attributable to the duration of the EGF treatment. In their experiments, EGF was applied for 7 days. The GLT-1 induction may be required for such prolonged EGF stimulation.
In some experimental models, GLAST appears to exhibit higher Km values than those of GLT-1, and is thus suspected of predominantly operating at pathologically elevated concentrations of glutamate (Kanai et al., 1997). It has already been reported that growth factors such as bFGF, IGF-1 and EGF prevent neurons from glutamate-mediated excitotoxicity (Abe and Saito, 1992; Maiese et al., 1993; Freese et al., 1992; Skaper et al., 1993; Nakao et al., 1996). Therefore, the protective effects of the growth factors are of interest with respect to glutamate transporter regulation. In light of our findings, the growth factors may facilitate clearance of glutamate under pathological conditions via GLAST upregulation. The observation that expression of bFGF increases following brain injury in vivo (Logan et al., 1992; Reilly and Kumari, 1996) raises the possibility that such protective mechanisms actually operate in the brain.
bFGF is known to bind to heparan sulfate proteoglycans at the cell surface and to the high-affinity FGF receptor, which contains a cytoplasmic tyrosine kinase domain (Givol and Yayon, 1992). bFGF receptor activation causes an increase in tyrosine kinase activity, and triggers phosphorylation of several proteins including the receptor itself, which is believed to be the initial step leading to biological actions of bFGF (Givol and Yayon, 1992). Similarly, IGF-1 and EGF bind and activate their receptor tyrosine kinases (Leof, 2000). Therefore it is possible that these growth factors stimulate a common signaling pathway to regulate GLAST expression. Indeed, the effects of all three growth factors were inhibited by herbimycin A, AFC, PD98059 or wortmannin. These results suggest that all these factors regulate GLAST expression via Ras/MAK and PI3K signaling cascades. However, these growth factors are unlikely to completely share the same signal transduction pathway.
A combination of IGF-1 with either bFGF or EGF resulted in a further increase in glutamate uptake activity, which strongly suggests that IGF-1 activates a different signaling pathway. This notion is further supported by a pharmacological study showing that genistein inhibited the effect of bFGF and EGF without affecting the effect of IGF-1. Genistein-sensitive and insensitive pathways may independently mediate the growth factor-induced regulation of GLAST expression. Interestingly, the effects of the growth factors were equally blocked by herbimycin A, a selective inhibitor of Src family kinases (Uehara et al., 1989), whereas genistein is known to be a relatively low selective inhibitor of protein tyrosine kinase (Akiyama et al., 1987; Uehara, 1997). Therefore, the tyrosine kinase involved specifically in IGF-1-activated signaling pathway cannot be deduced from our present data. A combination of bFGF and EGF had no additive effect on glutamate uptake activity, which suggests that bFGF and EGF may, at least in part, activate a common pathway. However, the effect of bFGF requires a [Ca2+]i rise, whereas the effect of EGF is dependent on PKC activation, but not vice versa. bFGF and EGF may use independent signaling pathways initially but converge at some later step in the induction of GLAST.
In conclusion, bFGF, IGF-1, and EGF modulate the expression of the glial Na+-dependent glutamate transporter through heterogenous signaling pathways. Currently, little is known about the promoter region of GLAST gene. Promoter analysis may elucidate these complicated regulations of GLAST by the growth factors. To our knowledge, our finding is the first evidence that glutamate transporters receive temporally tight regulation of gene expression (i.e. transient upregulation). The present study may provide a novel regulatory mechanism of glutamate transporters, and thus may be useful in exploring a new approach for preventing neurological diseases associated with glutamatergic neurotoxicity.
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ACKNOWLEDGMENTS |
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