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Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The Na+-dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short-term, as well as long-term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules present at the plasma membrane. A substrate-dependent change in the translocation rate of the transporter molecules accounts for the short-term effect, whereas the long-term modulation apparently involves transcriptional regulation. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors activation mediate a substantial reduction in the transcriptional activity of the chglast promoter through the Ca2+/diacylglicerol-dependent protein kinase (PKC) signaling cascade. Overexpression of constitutive active PKC isoforms of mimic the glutamate effect. Accordingly, increased levels of c-Jun or c-Fos, but not Jun-B, Jun-D or Fos-B, lower the chglast promoter activity. Serial deletions and electrophorectic mobility shift assays were used to define a specific region within the 5′ proximal region of the chglast promoter, associated with transcriptional repression. A putative glutamate response element could be defined in the proximal promoter stretch more likely between nts – 40 and − 78. These results demonstrate that GLAST is under glutamate-dependent transcriptional control through PKC, and support the notion of a pivotal role of this neurotransmitter in the regulation of its own removal from the synaptic cleft, thereby modulating, mainly in the long term, glutamatergic transmission.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate

AP-1

activator protein one

BGC

Bergmann glia cells

Bis I and Bis V

bisindolylmaleimide I or V

CAT

chloramphenicol acetyltransferase

chkbp

KBP gene

CREB

cyclic-AMP response-element binding protein

DMEM

Dulbecco's modified Eagle's medium

DNQX

6,7-dinitroquinoxaline

Glu

glutamate

HSV-TK

Herpes virus thymidine kinase

KA

kainic acid

KBP

kainate binding protein

PKC

protein kinase C

Sp1

stimulating protein one

TPA

12-tetradecanoyl-13-acetate YY-1, ying-yang transcription factor

Extracellular glutamate is normally kept at low levels by Na+ -dependent active transport systems expressed both in neurons and glial cells (Gegelashvili and Schousboe 1997). By these means, the concentration of this neurotransmitter is kept below toxic levels. Molecular cloning led to the isolation of three cDNAs that encode for different subtypes of high-affinity glutamate transporters systems: GLAST (Storck et al. 1992), GLT-1 (Pines et al. 1992), and EAAC1 (Kanai and Hediger 1992). Two additional amino acid transporters, EAAT4 and EAAT5, have been cloned to date (Fairman et al. 1995; Arriza et al. 1997). The bulk of extracellular glutamate is removed from the synaptic cleft by the astroglial transporters GLAST and GLT1, both proteins are expressed only in glial cells in the adult brain and in the spinal cord (Lehre et al. 1995; Schmitt et al. 1996, 1997). Recent experiments, using antisense oligonucleotides and targeted gene disruption have shown that GLAST and GLT-1 are critically involved in the neuronal protection against excitotoxicity in vivo. In fact, transporter levels change dramatically in disease states (Medina et al. 1996; Rothstein et al. 1996; Tanaka et al. 1997; Watase et al. 1998).

GLT-1 is expressed throughout the CNS, primarily in astrocytes, although it has also been reported to be present in a much lesser extent in neurons. In contrast, GLAST is expressed only in astrocytes throughout the CNS and is significantly enriched in the cerebellum (Shibata et al. 1997; Danbolt 2001). Within this structure, it has been localized in the molecular layer, specifically in a subset of glial cells, the Bergmann glia cells (BGC; Danbolt 2001). These cells are an excellent model in which glial–neuronal interactions can be analyzed. For instance, these cells express a repertoire of ionotropic and metabotropic glutamate receptors that changes as a result of glutamatergic stimulation. Moreover, their cell morphology and that of the Purkinje cells (which they surround) is also modified according to the expression of Ca2+-permeable iGluRs, suggesting a strong and continuous communication between these cell types (Watanabe 2002).

It has been documented that glutamate regulates GLAST activity, and most possibly membrane translocation, in these cells in a receptor-independent manner. Interestingly enough, this effect is protein kinase C (PKC)-dependent (González and Ortega 2000). Prolonged exposure to the PKC activator, 12-tetradecanoyl-13-acetate (TPA), results in a decrease in GLAST mRNA levels that suggest a transcriptional level of regulation as TPA does not modify GLAST mRNA half-life (Espinoza-Rojo et al. 2000). Indeed, when the activity of the chglast promoter was evaluated under glutamate exposure, a PKC-dependent reduction in activity was detected. This effect is mediated through Ca2+-permeable AMPA receptors (López-Bayghen et al. 2003a).

As a step forward in our understanding of GLAST transcriptional regulation, in this work we characterize the PKC isoforms involved in the glutamate effect and also delineate a region within the chglast promoter responsible for this regulation. Moreover, we characterize the appearance of differential DNA–protein complexes in this region as a result of AMPA receptors activation. These results might prove to be useful in the design of novel therapeutic strategies towards the treatment of neurodegenerative disorders in which GLAST is involved.

Materials

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Tissue culture reagents were obtained from Invitrogen (Gaithersburg, MD, USA). Glutamatergic agonists (glutamate, AMPA and antagonists (DNQX) were obtained from Tocris-Cookson (St Louis, MO, USA). Kainate was obtained from Ocean Produce International (Shelburne, Nova Scotia, Canada). [3H]-D-aspartate was obtained from New England Nuclear (Boston, MA, USA). Bisindolylmaleimide I and V and all other chemicals were from Sigma (St Louis, MO, USA) and Calbiochem (San Diego, CA, USA).

Cell culture and stimulation protocol

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Primary cultures of cerebellar BGC were prepared from 14-day-old chick embryos as described previously (Ortega et al. 1991). Cells were plated in 60-mm diameter plastic culture dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 2 mm glutamine, and gentamicin (50 µg/mL). Cells were used on the fourth or fifth day after culture. Before any treatment, confluent monolayers were switched to 0.5% serum medium and then treated as indicated. Antagonists or inhibitors were added 30 min before the agonists. Unless otherwise stated, the cells were treated with the excitatory amino acids analogs in culture medium for 2 h, this medium was replaced with DMEM containing 0.5% fetal bovine serum and the cells collected 48 h after in transfection experiments and 12 h after when the cells were processed for nuclear extracts preparation (see below).

Plasmids

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The plasmid p800GLASTCAT contains the 800-bp 5′-non-coding region from the chick GLAST gene (GenBank accession number: AY90600), cloned in the pCAT-BASIC vector (Promega, Madison, WI, USA) amplified by reverse PCR (López-Bayghen et al. 2003a). Serial deletions of p800GLASTCAT were obtained by enzymatic cuts in the appropriated sites (XbaI, ApaI, AccI, MaeIII) to generate D1, D2, D3, D4, and D5 constructs; exact positions for 5′ ends of this constructs are depicted in Fig. 5. Plasmids were sequenced using automated techniques. The reporter plasmid TRE-CAT was kindly donated by Michael Gredes from Dr Yuspa laboratory at NIH (Bethesda, MD, USA). TRE-CAT contains the structural gene for chloramphenicol acetyltransferase (CAT), under the control of the Herpes virus thymidine kinase (HSV-TK) promoter and five SV40 AP-1 sites cloned upstream (Rutberg et al. 1999). Dr Yuspa kindly donated the pSG5-based expression plasmids for c-jun, c-fos, jun-B, jun-D, and fos-B used in co-transfection experiments. The expression vectors encoding the constitutively active PKCα, -ɛ, -θ isoforms, and mutant PKC isoforms with a dominant negative (DN) (K[RIGHTWARDS ARROW]R) point mutation in the ATP binding site, have been described previously (Baier-Bitterlich et al. 1996; Soh et al. 1999; Soh and Weinstein 2003).

image

Figure 5. Deletion analysis of p800GLASTCAT. Left panel: Representation of the deletion constructs D1, D2, D3, D4, and D5 generated by restriction and cloning of resulted fragments in pCAT-BASIC vector (CAT reporter). BGC were transfected with complete or deletion constructs and 24 h post-transfection were treated with or without with 1 mm glutamate for 2 h. Results presented are the mean+-standard error of at least six independent experiments (*p < 0.001; anova).

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Transient transfections and CAT assays

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Transient transfections and CAT assays were performed in 60% confluent BGC cultures using calcium phosphate protocol with 3 µg of chglast reporter plasmid or 1 µg of TRE-CAT construct. Under such conditions, the transfection efficacy is ∼50% determined in every cell batch by an internal transfection control (β-gal). Treatment with glutamatergic ligands or inhibitors was performed 24 h post-transfection for the indicated time periods and concentrations. Protein lysates were obtained as follows: cells were harvested in TEN buffer (40 mm Tris–HCl pH 8.0, 1 mm EDTA, 15 mm NaCl), lysed with three freeze–thaw cycles in 0.25 m Tris–HCl pH 8.0 and centrifuged at 12 000 g for 3 min. Equal amounts of protein lysates (∼80 µg) were incubated with 0.25 µCi of [14C]-chloramphenicol (50 mCi/mmol, Amersham Biosciences, Piscataway, NJ, USA) and 0.8 mm Acetyl-CoA (Sigma) at 37°C. Acetylated forms were separated by thin-layer chromatography and quantified using a Typhoon Optical Scanner (Molecular Dynamics, Amersham Biosciences UK). CAT activities were expressed as the acetylated fraction corrected for the activity in the pCAT-BASIC vector and are expressed as relative activities to non-treated control cell lysates. Transient cotransfections for the expression of the full-length transcription factors or PKC isoforms were performed in BGC cultures using 1–3 µg of indicated plasmid DNA (construct expressing the protein or the empty vector). Co-transfection experiments were done under the same protocol stated before.

Electrophoretic mobility shift assays

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Nuclear extracts were prepared as described previously (López-Bayghen et al. 1996). All buffers contained a protease inhibitors cocktail to prevent nuclear factor proteolysis. Protein concentration was measured by the Bradford (1976) method. Nuclear extracts (approximately 5–7.5 µg) from control or agonist-treated BGC were incubated on ice with 500 ng of poly-[(dI-dC)] as non-specific competitor (Pharmacia) and 1 ng of [32P]-end-labeled double-stranded oligonucleotides: chGLAST/GLUr, 5′-TTTAAAACCATTTTCCCAAGTCCTGTCACTACATGCCCTT-3′; Sp1, 5′-CTAGATTCGATCGGGGCGGGGCGA-3′; AP-1, 5′-CTAGRATAATATGACTAAGCTGTG-3′.

The reaction mixtures were incubated for 20 min on ice, electrophoresed in 8% polyacrylamide gels using a low ionic strength 0.5X TBE buffer. The gels were dried and exposed to an autoradiographic film.

Transport assay in BGCs

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The uptake of [3H]-d-aspartate was performed on transfected cells as detailed elsewhere (Ruiz and Ortega 1995). Briefly, 24 h after transfection, the culture medium was exchanged with solution A (25 mm Hepes–Tris, 130 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl, 0.8 mm MgCl, 20 mm glucose and 1 mm NaHPO4, pH 7.4) and the cells pre-incubated for 30 min at 37°C. The cells were washed three times with solution A, and this medium exchanged for solution A containing [3H]-d-aspartate (0.4 mCi/mL) and incubated for 10 min. Thereafter, the medium was removed by rapid aspiration, then monolayers were washed with ice-cold solution A and were used for protein determination and liquid scintillation counting. All the uptake data were analyzed using the SIGMA PLOT 2000 software program (Sysat Software Inc., Point Richmond, CA, USA).

Statistical analysis

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Data are expressed as the mean ± SE. A one-way analysis of variance (anova) was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post-hoc Student–Newman–Keuls test analysis was used to determine which conditions were significantly different from each other (Prism, GraphPad Software, San Diego, CA, USA).

Glutamate exposure decreases GLAST promoter activity via PKC

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The activity of the chglast promoter is regulated by glutamate treatment in BGC through AMPA receptors in a Ca2+-dependent manner (López-Bayghen et al. 2003a). Moreover, the PKC activator phorbol, TPA, drives a significant reduction in GLAST transporter activity (González and Ortega 2000) and lowers its mRNA levels, without affecting its half-life (Espinoza-Rojo et al. 2000). Taking into consideration that AMPA receptors activation results in an activation of PKC in BGC (Cid and Ortega 1993), we decided to evaluate the involvement of PKC in the regulation of chglast promoter activity elicited by glutamate receptors activation. In line with our preliminary findings, a significant reduction in promoter activity is obtained after a 2 h exposure to 1 mm glutamate or 100 nm TPA (Fig. 1). The PKC inhibitors BisI or staurosporine block both effects. In order to rule out an unspecific effect, we tested the integrity of the PKC signaling cascade after glutamate treatment via a TRE-CAT construct (Fig. 1b). In this plasmid, the HSV-TK promoter is directed by five SV40 AP-1 sites (Fig. 1b). As expected, glutamate and TPA treatment increased the promoter activity, in accordance with the increase in AP-1 binding to SV40 AP-1 sites that we reported before (Aguirre et al. 2000).

image

Figure 1. The chglast promoter is downregulated by glutamate via PKC. (a) Schematic representation of the 5′-non-coding region of the GLAST putative promoter region (− 515 to + 248, total 763 bp) cloned in front of the CAT reporter gene (p800GLASTCAT). Transcription factor binding sites are denoted. Arrow indicates the putative TATA box. BGC were transfected with this construct and the activity relative to the non-treated control (N/S) was obtained. Cells were treated 24 h post-transfection for 2 h with glutamate (1 mm); the PKC inhibitors, staurosporine (500 nm, ST) and BisI (1 µm) were added 30 min before glutamate or TPA. The non-active form of BisI (BisV, 1 µm) was also added before glutamate (GLU) or TPA. Cells were harvested 48 h after transfection and processed for CAT activity assays. (b) Map and activity of TRE-CAT construct containing five AP-1 sites cloned in front of the HSV Timidine Kinase promoter and CAT reporter gene. Results presented are the mean+-standard error of at least six independent experiments (*p < 0.001; vs. untreated, by anova).

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Overexpression of PKC isoforms mimic the glutamate transcriptional effect

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The complexity of PKC signaling has been emphasized by molecular cloning and sequencing which revealed that PKC exists as a family of enzymes: the classical subtypes (α, βΙ,ΙΙ, γ) that are activated by phorbol esters and are Ca2+-dependent, the novel subtypes (δ, ε, η, θ), which are also activated by phorbol esters but are Ca2+-independent, and the atypical isoforms (ζ, λ), which are not activated by phorbol esters (Kazanietz 2002).

In order to gain insight into the PKC isoforms relevant for GLAST transcriptional regulation, we overexpressed several constitutive active PKC isoforms. Although a detailed description of the PKC isoforms that are expressed in chick Bergmann glia is not available, a developmental study of the different PKC isoforms present in chick embryo CNS has been reported (Mangoura et al. 1993). Worth mentioning is the fact that these authors find the expression of PKCε and PKCα in cerebellar tissue. As depicted in Fig. 2, overexpression of constitutive active PKCα or PKCε significantly reduced chglast promoter activity. In contrast, increased levels of an active form of PKCθ do not modify the promoter activity. As expected, overexpression of negative dominant mutants of these PKC isoforms have no effect. These results suggest, on the one hand, that the glutamate effect is carried out by more than one signaling pathway (PKC overexpression is less effective than the glutamate treatment) and, on the other hand, that both classical as well as novel PKC isoforms might be involved in this phenomena. Transfection of the atypical PKCγ does not change CAT activity (data not shown). In order to validate these results, we measured the transcriptional activity of the bona fide PKC-dependent promoter, the TRE-CAT construct and, as shown in Fig. 2(b), a strong induction of the activity of the TRE-CAT construct is obtained after an increased expression of PKCα or -θ isoforms. In other to support further the involvement of a particular PKC isoform in the glutamate effect, we co-transfected increasing concentrations of the dominant negative PKC isoforms (α, ε, θ) with the chglast reporter plasmid and exposed these cells to 1 mm glutamate for 2 h. The results are shown in Fig. 2(c). Note that only the dominant negative α isoform is capable of reverting the glutamate effect, whereas only a partial reversion is obtained with 3 µg of the PKCε plasmid. These results suggest a major role for PKCα in the glutamate-mediated transcriptional repression of the chglast promoter, although it is clear that PKCε shares substrates relevant for the transcriptional response with PKCα because when it is overexpressed in its active form it reduces the promoter activity as glutamate (albeit to a lower extent). Besides, its dominant negative mutant decreases by 50% the glutamate effect when a huge amount of the inactive kinase is present (Fig. 2). As expected, the promoter activity of the TRE-CAT construct upon glutamate exposure is reduced with either of the three dominant negative constructs (Fig. 2d).

image

Figure 2. Differential PKC isoforms are involved in the glutamate effect. Cells were cotransfected with constructs overexpressing different PKC isoforms, PKC mutant isoforms or empty vector (Vect) along the p800GLASTCAT reporter construct (chglast). GLU (1 mm, 2 h) or TPA (100 nm, 2 h) treatments were applied 24 h post-transfection when indicated. TRE-CAT construct was co-transfected in same conditions with the indicated PKC isoforms. Triangle indicates increasing amounts of constructs expressing PKC mutant isoforms (from 1 to 3 µg). Results are the mean ± SE of at least six independent experiments. (*p < 0.001 vs. untreated, by anova).

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Fos and Jun participate in chglast promoter activity regulation

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Previous results from our group have shown the glutamate treatment of BGC results in an induction of the activator protein-1 (AP-1) DNA binding activity. Furthermore, when two components of this transcription factor, such as c-Fos and c-Jun, are overexpressed in BGC, a substantial increase in the chick kainate binding protein promoter (chkbp) activity is obtained (Aguirre et al. 2000). Taking into consideration that our positive control, the TRE-CAT construct, is driven by five AP-1 sites, we decided to evaluate the involvement of several members of the Fos and Jun families over the activity of the chglast promoter. The results are depicted in Fig. 3, co-transfection of chglast with c-fos, c-jun, and, to a lesser extent, fos-B resulted in a negative regulation of the chglast construct (Fig. 3). Neither jun-B nor jun-D changed the promoter activity. Note that jun-D is capable of inducing the promoter activity of the TRE-CAT construct.

image

Figure 3. Increase in c-Jun or c-Fos levels downregulate chglast promoter activity. BGC were co-transfected with constructs overexpressing Jun and Fos proteins as indicated or the pSG5 empty vector (VECT, 1 µg each case) and the p800GLASTCAT construct (chglast). Results are the mean ± SE of at least six independent experiments (*p < 0.001; **p < 0.05 vs. untreated, by anova).

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[3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

The results described thus far provide evidence for a PKC-dependent modulation of chglast transcription. Besides, we have already shown that the PKC activator, TPA reduces not only the GLAST-mediated transport in BGC, but also its protein levels (González and Ortega 2000). Taking into consideration that biotinylation experiments have demonstrated that the levels of GLAST in the plasma membrane are also regulated (Robinson 2003), we decided to explore if the transcriptional effect described could actually affect the transport. The results are presented in Fig. 4: [3H]-d-aspartate uptake is significantly reduced when BGC are transfected with the plasmids that encode for PKCα, PKCε, c-fos, and c-jun. As it has been clearly documented, treatment of BGC with glutamate (1.0 mm) for 30 min, results in a substantial reduction of [3H]-d-aspartate uptake that is related to an increase in the transporter's Km (González and Ortega 2000). These results therefore confirm that, despite the existence of other levels of regulation, in the long term GLAST mRNA diminished levels are reflected in a reduced glutamate transport.

image

Figure 4. Effect of overexpression of different PKC isoforms and AP- 1 components in [3H]-d-aspartate uptake in Bergmann glia cells. Cells were transfected with the indicated plamids and 48 h post-transfection were used for uptake studies. Mock-transfected cells were incubated with 1.0 mm of glutamate at room temperature (24°C) for 30 min before the transport assay (used as a control). Subsequently, cells were washed with solution A, and immediately [3H]- d-aspartate uptake was performed (0.4 mCi/mL, for 10 min). Values represent triplicate determinations and are the mean ± SE of six independent experiments. Data were compared by anova (*p < 0.001; **p < 0.05; ***p < 0.01).

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Mapping of the glutamate response fragment in the chglast promoter

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

At this stage, a deletion analysis over the chglast promoter was imperative in order to map the region involved in the glutamate effect. Five new constructs were generated and tested by transfection assays in BGC (Fig. 5). Activity of the various deletion constructs is expressed relative to the chglast promoter. Note that the removal of the fragment comprised between nts − 40 and − 78 (constructs D3 and D5) abolishes the glutamate-mediated reduction in transcriptional activity. It is worth mentioning that other negative regulatory elements are predicted to be present with in the region comprised between nts – 78 and −214 as the promoter activity of constructs D1 and D2 are significantly lower than that of construct D4.

A pharmacological and signaling profile of the glutamate effect was established using the non-responsive construct D3 or D4 construct, which contains up to the glutamate response region. As expected, the pharmacological tools used were ineffective in modifying the D3 transcriptional activity (Fig. 6). In sharp contrast, when the activity of the D4 was assayed in cells pre-incubated with the AMPA receptors antagonist 6,7-dinitroquinoxaline (DNQX) or the PKC inhibitor BisI and exposed to 1 mm glutamate, the negative effect of this amino acid is completely reversed. In line with these results, TPA treatment leads to a substantial reduction in promoter activity (Fig. 6). Taken together, these data demonstrate that glutamate reduces chglast promoter activity via PKC and a cis element located most probably in the region between nts − 40 and − 78, which we termed chGLAST/GLUr.

image

Figure 6. PKC regulates the activity of the D4 construct. Left panel: Representation of D3 and D4 constructs transfected into BGC. After a 24-h period, cells were treated for 2 h with 1 mm glutamate; 100 nm TPA. The AMPA receptors antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 100 µm), the PKC inhibitors, staurosporine (500 nm, ST) and BisI (1 µm) and its inactive analogue BisV were added 30 min before Glu or TPA. Results presented are the mean ± SE of at least six independent experiments (*p < 0.001; anova).

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DNA–protein complexes in the glutamate-responsive fragment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

In accordance with the transcriptional response to glutamate obtained by deletion analysis, the chGLAST/GLUr may be responsible for the negative transcriptional response. In order to gain insight into this, we decided to detect DNA–protein interactions within this region. An oligonucleotide containing this sequence was labeled and tested with nuclear extracts obtained from BGC exposed to glutamate (Fig. 7). A time- and dose-dependent appearance of two DNA–protein complexes, named X and Y, was detected. Changes in the complex pattern can be easily noticed when the glutamate concentration is higher than 10 µm and after 30 min of treatment (Fig. 7a). Interestingly enough, DNQX is able to inhibit the formation of these two complexes upon glutamate treatment, but not after TPA exposure (Fig. 7b). The PKC blockers, BisI and staurosporine, reverted the TPA effect, as expected. Competition experiments were performed in order to try to identify these complexes, an obvious candidate was AP-1, because a putative binding sequence for this factor is present in the region necessary for glutamate repression (Fig. 4). Surprisingly, a canonical AP-1 site such as that present in SV-40 did not displace the X or Y complexes (competition experiment, Fig. 7d), suggesting that the identity of retarded bands is other than AP-1. In order to rule out any technical problem with the AP-1 probe, we measured, in these extracts, the DNA–protein complexes formed with this SV40 oligonucleotide probe radiolabeled and, as expected, we were able to detect the characteristic glutamate-induced AP-1 complex. In the same vein, note that neither glutamate nor TPA exposure modify the complex formed with an Sp1 oligonucleotide (Fig. 7b). The identity of the X and Y complexes is not known at this moment and their characterization is beyond the scope of this paper. Nevertheless, additional computer analysis over the sequence comprised in the chGLAST/GLUr is presented in Fig. 8, in which we have underlined different elements that can be recognized by other transcription factors. Current experiments are underway in our lab in order to define the important trans factors involved in chglast promoter regulation.

image

Figure 7. Glutamate-dependent DNA–protein interactions in the chGLAST/GLUr element. Nuclear extracts were prepared from control or glutamate-treated (1 mm) BGC for indicated time periods or for 2 h with indicated glutamate concentrations. Gel shift assays were performed using as [32P]-end-labeled oligonucleotides: AP-1 (AP-1 site from SV40 virus), Sp1 consensus sequence (1 ng each) or the 40-bp fragment chGLAST/GLUr (see Fig. 8a). (a) Comparison of DNA binding activity between nuclear extracts from control (N/S, non-stimulated) or glutamate-stimulated (GLU, 1 mm 2 h) BGC for the indicated time periods or for 2 h using increasing glutamate concentrations (1–1000 µm) using different probes. Black arrows indicate the complexes that change upon after stimulation. (b)  chGLAST/GLUr was tested with nuclear extracts from BGC treated for 2 h with 1 mm glutamate; 100 nm TPA, AMPA, 1 mm KA, AMPA receptors antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 100 µm), the PKC inhibitors, staurosporine (500 nm, ST), and BisI (1 µm) and its inactive analogue BisV (1 µm). Inhibitors were added 30 min before GLU, AMPA, KA or TPA and cells harvested after a 12-h period. (c) The same nuclear extracts as in (b) were used to detect the Sp1 characteristic complexes (Sp1cx). (d) Competition experiments, competitors were added in the 50-, 150-, and 300-fold excess. (e) The AP-1cx detected with AP-1 SV 40 probe tested against same extracts indicated in panel B. All results shown are representative of at least three independent experiments with similar results.

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image

Figure 8. Extended sequence analysis of chglast promoter. (a) Nucleotide sequence of the chglast promoter region. Underlined are the putative binding sites for the indicated transcription factors obtained using Genomatix Software GmbH 1998–2004 (http://www.genomatix.de/).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References

Glutamate transporters play a pivotal role in the control of the extracellular levels of this neurotransmitter. Several regulatory mechanisms modulate the uptake process, both acutely as well as chronically. The protection of neuronal cells from an excitotoxic insult that can lead to neuronal death relies on these regulatory processes. Evidently, the establishment of these biochemical transactions will be a valuable tool in the development of new strategies against the various neurodegenerative disorders in which glutamate is involved.

The results presented in this work demonstrate that PKC is involved in the transcriptional regulation of the chglast promoter upon the activation of AMPA receptors. Moreover, a putative glutamate response element (chGLAST/GLUr) was mapped. Conflicting results have been reported concerning the effect of PKC activation on the transport activity of GLAST (Robinson 2003). It has been hypothesized that these differences are related to the specific cellular milieu, meaning that different proteins are required for the trafficking of GLAST in the various cultures examined, or that different PKC isoforms are expressed in these cultured cells (Susarla and Robinson 2003). Therefore, we decided to take a different approach: we expressed different PKC isoforms in BGC, asked ourselves whether these isoforms had the same effect on the chglast transcriptional activity, and if these changes would affect the transport of [3H]-d-aspartate.

We were able to demonstrate that the expression of either a classical (α) or a novel isoform (ε) downregulates the promoter activity as well as the uptake (Figs 2 and 4). It should be noted, however, that these results do not demonstrate that the referred PKC isoforms are components of the glutamate signaling cascade. In fact, when the dominant negative versions of these kinases were transfected and the glutamate effect determined, only the α isoform reverted completely the agonist effect (Fig. 2c). This interpretation is in line with our previous findings concerning the strict dependence on external Ca2+ of the glutamate effect (López-Bayghen et al. 2003a). Most probably, PKCε when overexpressed, is phosphorylating a PKCα target involved in the chglast transcriptional regulation. In this context, it is not surprising the poor capacity of the dominant negative version of PKCε to block the glutamate effect (Fig. 2c).

It is evident that other signaling cascades are involved in the glutamate effect as neither of the transfected isoforms are as effective as the exposure to the excitatory amino acid. In this regard, we have previously reported the involvement of tyrosine kinases cascades in the transcriptional regulation of the chkbp promoter through AMPA receptors (López-Bayghen et al. 2003b). In any event, when we evaluated the role of a downstream element of glutamate signaling such as the components of the AP-1 transcription factors, we were capable to mimic quantitatively the glutamate effect by overexpressing either c-Fos or c-Jun (Figs 2 and 4), suggesting that whatever are the signaling cascades activated by glutamate, they converge at the level of the expression of AP-1.

At this stage, we decided to generate five different constructs in order to delineate the cis acting elements in the chglast promoter relevant to its negative regulation. To our surprise, the predicted AP-1 sites (Figs 1 and 8) are not involved in the transcriptional effect (Fig. 7). One could argue that these AP-1 sites lie inside the coding region, although the functionality of an AP-1 site within exon 1 of the propiomelanocortin gene has been reported (Boutillier et al. 1995). A discrete region comprising nts – 40 to −78 is definitely involved, although other putative negative regulators should be present in the region from nts − 78 to − 214, as the promoter activities in constructs D1 and D2 are lowered more efficiently by glutamate than the activity in construct D4 (Fig. 5).

A gel-shift strategy was undertaken in order to characterize the chGLAST/GLUr region. Two major complexes, which we termed X and Y, were detected as a function of the time and dose of exposure of BGC to glutamate (Fig. 7). Note that TPA overrides the blockage of AMPA receptors with DNQX. As predicted from the results obtained in reporter gene assays, the formation of these complexes is insensitive to the competition with a 100-fold excess of an unlabeled AP-1 double-stranded oligomer. Computer-based analysis of what we termed chGLAST/GLUr revealed overlapping YY-1 and Ikaros binding sites. YY-1 is a transcription factor with dual functionality, although it is well established that it represses transcription by physically interacting with the general transcription machinery (Ogbourne and Antalis 1998). The close proximity of this sequence to the TATA box could favor a role for YY-1 in chglast transcriptional downregulation. Concerning the Ikaros proteins, these are characterized by zinc finger domains that can activate or repress transcription either by binding to its consensus C/TGGGAA/T sequence or through its interaction with chromatin remodeling complexes (Rebollo and Schmitt 2003). Whatever the identity of the X and Y complexes may be, it is evident that c-Fos and/or c-Jun influence their interaction with chGLAST/GLUr, either by leading to their synthesis or by protein–protein interactions. A current model for the findings described thus far is presented in Fig. 9.

image

Figure 9. Current model for chglast transcriptional activation in BGC. Increased extracellular glutamate levels activate AMPA receptors leading to Ca2+ entrance. Activation of Ca2+-dependent phospholipase C results in increased diacylglycerol levels that activate primarily PKCα and probably PKCε (dashed lines). These enzymes are involved in the increase in AP-1 DNA binding which in turn presumably regulate the synthesis of a repressor protein(s) that interact with the chGLAST/GLUr downregulating its transcription. Extracellular glutamate is taken up by GLAST, which by an unidentified mechanism activates PKC, and could in principle regulate its own transcription.

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In summary, we provide here evidence for the involvement of PKCα in the glutamate-dependent transcriptional regulation of GLAST. Moreover, we define a 38-nt region that reduces chglast transcription. Work currently underway in our laboratory is aiming to characterize of the transcription factors that interact with the chGLAST/GLUr.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Plasmids
  7. Transient transfections and CAT assays
  8. Electrophoretic mobility shift assays
  9. Transport assay in BGCs
  10. Statistical analysis
  11. Results
  12. Glutamate exposure decreases GLAST promoter activity via PKC
  13. Overexpression of PKC isoforms mimic the glutamate transcriptional effect
  14. Fos and Jun participate in chglast promoter activity regulation
  15. [3H]-d-aspartate uptake modulation by PKC, c-Fos, and c-Jun
  16. Mapping of the glutamate response fragment in the chglast promoter
  17. DNA–protein complexes in the glutamate-responsive fragment
  18. Discussion
  19. Acknowledgements
  20. References
  • Aguirre A., Lopez T., Lopez-Bayghen E. and Ortega A. (2000) Glutamate regulates kainate-binding protein expression in cultured chick Bergmann glia through an activator protein-1 binding site. J. Biol. Chem. 275, 3924639253.DOI: 10.1074/jbc.M002847200
  • Arriza J. L., Eliasof S., Kavanaugh M. P. and Amara S. G. (1997) Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl Acad. Sci. USA 94, 41554160.DOI: 10.1073/pnas.94.8.4155
  • Baier-Bitterlich G., Uberall F., Bauer B., Fresser F., Wachter H., Grunicke H., Utermann G., Altman A. and Baier G. (1996) Protein kinase C-τ isoenzyme slictive stimulation of the transcription factor complex AP-1 en T-lymphocytes. Mol. Cell Biol. 16, 18421850.
  • Boutillier A. L., Monnier D., Lorang D., Lundblad J. R., Roberts J. L. and Loeffler J. P. (1995) Corticotropin-releasing hormone stimulates proopiomelanocortin transcription by cFos-dependent and -independent pathways: characterization of an AP1 site in exon 1. Mol. Endocrinol. 9, 745755.
  • Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254.
  • Cid M. E. and Ortega A. (1993) Glutamate stimulates [3H]phorbol 12,13-dibutyrate binding in cultured Bergmann glia cells. Eur. J. Pharmacol. 245, 5154.
  • Danbolt N. C. (2001) Glutamate uptake. Prog. Neurobiol. 65, 1105.DOI: 10.1016/S0301-0082(00)00067-8
  • Espinoza-Rojo M., López-Bayghen E. and Ortega A. (2000) GLAST: gene expression regulation by phorbol esters. Neuroreport 11, 28272832.
  • Fairman W. A., Vandenberg R. J., Arriza J. L., Kavanaugh M. P. and Amara S. G. (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375, 599603.DOI: 10.1038/375599a0
  • Gegelashvili G. and Schousboe A. (1997) High-affinity glutamate transporters: regulation of expression and activity. Mol. Pharmacol. 52, 615.
  • González M. I. and Ortega A. (2000) Regulation of high-affinity glutamate uptake activity in Bergmann glia cells by glutamate. Brain Res. 866, 7381.DOI: 10.1016/S0006-8993(00)02226-5
  • Kanai Y. and Hediger M. A. (1992) Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360, 467471.DOI: 10.1038/360467a0
  • Kazanietz M. G. (2002) Novel ‘nonkinase’ phorbol ester receptors: the C1 domain connection. Mol. Pharmacol. 61, 759767.DOI: 10.1124/mol.61.4.759
  • Lehre K. P., Levy L. M., Ottersen O. P., Storm-Mathisen J. and Danbolt N. C. (1995) Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J. Neurosci. 15, 18351853.
  • López-Bayghen E., Vega A., Cadena A., Granados S. E., Jave L. F., Gariglio P. and Alvarez-Salas L. M. (1996) Transcriptional analysis of the 5′-noncoding region of the human involucrin gene. J. Biol. Chem. 271, 512520.DOI: 10.1074/jbc.271.1.512
  • López-Bayghen E., Aguirre A. and Ortega A. (2003b) Transcriptional regulation through glutamate receptors: Involvement of tyrosine kinases. J. Neurosci. Res. 74, 717725.DOI: 10.1002/jnr.10807
  • López-Bayghen E., Espinoza-Rojo M. and Ortega A. (2003a) Glutamate downregulates GLAST expression through AMPA receptors in Bergmann glial cells. Brain Res. Mol. Brain Res. 115, 19.
  • Mangoura D., Sogos V. and Dawson G. (1993) Protein kinase c-epsilon is a developmentally regulated, neuroanl isoform in the chick central nervous system. J. Neurosci. Res. 35, 488498.
  • Medina L., Figueredo-Cardenas G., Rothstein J. D. and Reiner A. (1996) Differential abundance of glutamate transporter subtypes in amyotrophic lateral sclerosis (ALS)-vulnerable versus ALS-resistant brain stem motor cell groups. Exp. Neurol. 142, 287295.DOI: 10.1006/exnr.1996.0198
  • Ogbourne S. and Antalis T. M. (1998) Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem. J. 331, 114.
  • Ortega A., Eshhar N. and Teichberg V. I. (1991) Properties of kainate receptor/channels on cultured Bergmann glia. Neuroscience 41, 335349.DOI: 10.1016/0306-4522(91)90331-H
  • Pines G., Danbolt N. C., Bjoras M. et al. (1992) Cloning and expression of a rat brain 1-glutamate transporter. Nature 360, 464467.DOI: 10.1038/360464a0
  • Rebollo A. and Schmitt C. (2003) Ikaros, Aiolos and Helios: transcription regulators and lymphoid malignancies. Immunol. Cell Biol. 81, 171175.DOI: 10.1046/j.1440-1711.2003.01159.x
    Direct Link:
  • Robinson M. B. (2003) Signaling pathways take aim at neurotransmitter transporters. Sci. STKE 2003: pe 50.
  • Rothstein J. D., Dykes-Hoberg M., Pardo C. A. et al. (1996) Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675686.DOI: 10.1016/S0896-6273(00)80086-0
  • Ruiz M. and Ortega A. (1995) Characterization of an Na(+)-dependent glutamate/aspartate transporter from cultured Bergmann glia. Neuroreport 6, 20412044.
  • Rutberg S. E., Adams T. L., Olive M., Alexander N., Vinson C. and Yuspa S. H. (1999) CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes. Oncogene 18, 15691579.DOI: 10.1038/sj.onc.1202463
  • Schmitt A., Asan E., Puschel B., Jons T. and Kugler P. (1996) Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry. Neuroscience 71, 9891004.DOI: 10.1016/0306-4522(95)00477-7
  • Schmitt A., Asan E., Puschel B. and Kugler P. (1997) Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry. J. Neurosci. 17, 110.
  • Shibata T., Yamada K., Watanabe M., Ikenaka K., Wada K., Tanaka K. and Inoue Y. (1997) Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord. J. Neurosci. 17, 92129219.
  • Soh J. W. and Weinstein I. B. (2003) Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J. Biol. Chem. 278, 3470934716.DOI: 10.1074/jbc.M302016200
  • Soh J. W., Lee E. H., Prywes R. and Weinstein I. B. (1999) Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol. 19, 13131324.
  • Storck T., Schulte S., Hofmann K. and Stoffel W. (1992) Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc. Natl Acad. Sci. USA 89, 1095510959.
  • Susarla B. T. and Robinson M. B. (2003) Rottlerin, an inhibitor of protein kinase C delta (PKCΔ), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCΔ-independent. J. Neurochem. 86, 635645.DOI: 10.1046/j.1471-4159.2003.01886.x
  • Tanaka S., Kiuchi Y., Numazawa S., Oguchi K., Yoshida T. and Kuroiwa Y. (1997) Changes in glutamate receptors, c-fos mRNA expression and activator protein-1 (AP-1) DNA binding activity in the brain of phenobarbital-dependent and -withdrawn rats. Brain Res. 756, 3545.DOI: 10.1016/S0006-8993(97)00134-0
  • Watanabe M. (2002) Glial processes are glued to synapses via Ca(2+)-permeable glutamate receptors. Trends Neurosci. 25, 56.DOI: 10.1016/S0166-2236(00)01993-7
  • Watase K., Hashimoto K., Kano M. et al. (1998) Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur. J. Neurosci. 10, 976988.DOI: 10.1046/j.1460-9568.1998.00108.x