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Keywords:

  • glia;
  • glutamate receptors;
  • transcriptional regulation

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Ionotropic glutamate receptors in cerebellar Bergmann glial cells are linked to transcriptional regulation and, by these means, are thought to play an important role in plasticity, learning and memory and in several neuropathologies. Within the CNS, the transcription factors of the POU family bind their target DNA sequences after a growth factor-dependent phosphorylation–dephosphorylation cascade. Exposure of cultured Bergmann glial cells to glutamate leads to a time- and dose-dependent increase in Oct-2 DNA-binding activity. The use of specific pharmacological tools established the involvement of Ca2+-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. Furthermore, the signaling cascade includes phosphatidyl inositol 3-kinase as well as protein kinase C activation. Interestingly, transcriptional as well as translational inhibitors abolish the glutamate effect, suggesting a transcriptional up-regulation of the oct-2 gene. These data demonstrate that Oct-2 expression is not restricted to neurons and further strengthen the notion that the glial glutamate receptors participate in the modulation of glutamatergic cerebellar neurotransmission.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methylisoxazole-4-propionate

BGC

Bergmann glial cell

Glu

glutamate

iGluR

ionotropic glutamate receptors

KBP

kainate-binding protein

PBS

phosphate-buffered saline

PKC

protein kinase C

sil2cx

SIL-2 complex

TPA

12-O-tetradecanoylphorbol-13-acetate

Glutamate (Glu) is the main excitatory neurotransmitter in the CNS and it exerts its action via specific membrane receptors. Glutamate receptors are classified in two groups, ligand-gated ion channels (iGluRs) and metabotropic receptors coupled to G proteins. The iGluRs contain integral, cation-specific ion channels and have been subdivided into NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainic acid receptors (Gasic and Hollmann 1992; Hollmann and Heinemann 1994). In contrast, metabotropic Glu receptors are subdivided into three subgroups that are preferentially activated by quisqualate, ibotenate and 1-amino-4,5-cyclopentane-trans-1,3-dicarboxylate, respectively (Pin and Duvoisin 1995).

Bergmann glial cells (BGCs) carry, in their membranes, AMPA, NMDA and metabotropic receptors (Lopez et al. 1994, 1997, 1998). The activation of these receptors induces, among other signaling events, Ca2+ influx, phosphoinositide hydrolysis, protein kinase C (PKC) translocation to the membranes, activation of the MAPK pathways, activation of phosphatidyl inositol 3-kinase and activation of inducible transcription factors (Cid and Ortega 1993; Sanchez and Ortega 1994; Lopez-Colome et al. 1997; Aguilera and Ortega 1999; Aguirre et al. 2000; Iino et al. 2001; Gamboa and Ortega 2002). Kainate-binding proteins (KBPs) are members of the iGluR gene family (Henley 1994). In the chick, KBP expression is restricted to BGCs in the cerebellum (Somogyi et al. 1990; Ortega et al. 1991), matches the time of granular cell migration (Henley 1994) and, interestingly, is also up-regulated by an imprinting stimulus in ducks (Kimura et al. 1993).

In the cerebellum, BGCs establish an intimate relationship with the glutamatergic Purkinje cell–parallel fiber synapse. Exposure of cultured BGCs to Glu leads to an increase in KBP and its mRNA levels through an activator protein 1-binding site, although a putative negative regulatory element in the −250/−170 region has also been defined (Aguirre et al. 2000).

The octamer motif (ATGCAAAT) is found in the promoters and enhancers of a number of different genes, such as those encoding the small nuclear RNAs, histone H2B, the immunoglobulin light chains and, interestingly, in KBP. The different effects of the octamer motif in different promoters and cell types are likely to be determined by the nature of the octamer-binding protein present in each cell type. Thus, the B cell-specific expression of the immunoglobulin genes is associated with the presence in these cells of the octamer-binding protein Oct-2 (Latchman 1999). Conversely, the octamer-dependent inhibition of the immunoglobulin enhancer in embryonal carcinoma cells is due to the expression of Oct-2 in these cells (Latchman 1996). Interestingly, in neuronal cells, Oct-2 acts as a transcriptional inhibitor and has been shown to be responsible for the repression of HSV immediate–early gene expression (Thomas et al. 1998). Hence, the Oct-2 protein can act as a transcriptional activator or repressor depending on the cell type examined.

Although Oct-2 expression within the CNS is apparently confined to neurons, the fact that an octamer-related sequence is present in a putative negative regulatory region of a glial-specific protein, KBP, prompted us to investigate the presence of Oct-2 in BGCs. Not only were we able to identify this protein in glial cells but we also demonstrate here that its protein levels and DNA-binding activity are induced through AMPA receptor activation. These observations suggest that Oct-2 participates in Glu-dependent transcriptional regulation and add another level of complexity to Glu receptor signaling transactions.

Materials

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Tissue culture reagents were obtained from Gibco (Gaithersburg, MD, USA). Glutamatergic agonists (Glu, quisqualate, 1-amino-4,5-cyclopentane-trans-1,3-dicarboxylate, LAP-4, NMDA and AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione) were obtained from Tocris Cookson (St Louis, MO, USA). Kainate was obtained from Ocean Produce (Shelborne, Nova Scotia, Canada). Protease inhibitors were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). The antibodies used were rabbit polyclonal anti-KBP, rabbit polyclonal anti-Oct-1 sc-232, rabbit polyclonal anti-Oct-2 sc-233 and rabbit polyclonal anti-p53 sc-6243 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-linked anti-mouse or anti-rabbit immunoglobulins and the enhanced chemiluminescence reagent were obtained from Amersham (Little Chalfont, Buckinghamshire, UK). Biotinylated goat anti-rabbit serum and avidin-biotin-horseradish peroxidase were from Vector Laboratories (Burlingame, CA, USA). The Diamond phosphoprotein gel stain was obtained from Molecular Probes (Eugene, OR, USA). All other chemicals were from Sigma (St Louis, MO, USA).

Cell culture and stimulation protocol

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Primary cultures of cerebellar BGCs were prepared from 14-day-old chick embryos as described previously (Ortega et al. 1991). Cells were plated in 100-mm diameter plastic culture dishes in Dulbecco's modified Eagle's medium 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 incubated for 7 h in 0.5% serum medium and then treated as indicated. Antagonists or inhibitors were added 30 min before the agonists. CEC-32 chick fibroblasts (Kaaden et al. 1982), HeLa and HL60 cell lines were grown under the same media conditions used for BGCs. Unless stated otherwise, the cells were treated with the excitatory amino acid analogs in culture medium for 1 h, this medium was replaced with Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and the cells collected 3 h later and processed for nuclear extract preparation (see below). Antagonists were added 30 min before antagonists.

Electrophoretic mobility shift assays

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Nuclear extracts were prepared as described previously (Lopez-Bayghen et al. 1996). All buffers contained a protease inhibitor cocktail to prevent nuclear factor proteolysis. Protein concentration was measured by the Bradford method (Bradford 1976). Nuclear extracts (approximately 5–15 µg) from control or agonist-treated BGCs were incubated on ice with 1 µg of poly[(dI-dC)] as non-specific competitor (Amersham) and 1 ng of [32P] end-labeled double-stranded oligonucleotides: SIL-2, 5′-AGC TTT ATC TGT ATT TTC CGA GTC-3′; Oct-1, 5′-TGT CGA ATG CAA ATC ACT AGA A-3′; OctM, 5′-TGT CGA ATG CAA GCC ACT AGA A-3′ and stimulating protein 1, 5′-CTAGATTCGATCGGGGCGGGGCGA-3.

The reaction mixtures were incubated for 10 min on ice and electrophoresed in 7.8% polyacrylamide gels using a low ionic strength 0.5× TBE buffer. The gels were dried and exposed to an autoradiographic film or scanned with a Typhoon Optical Scanner (Molecular Dynamics, Sunnyvale, CA, USA).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

The cells from confluent monolayers were harvested and washed several times with phosphate-buffered saline (PBS) (10 mm K2HPO4/KH2PO4, 150 mm NaCl, pH 7.4). Cells were lysed with 50 mm Tris-HCl, pH 7.5, with protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 1 mg/mL aprotinin and 1 mg/mL leupepetin); aliquots of this suspension were used for protein concentration determination and boiled for 5 min in Laemmli's sample buffer. Equal amounts of protein (approximately 80 µg/lane) were resolved through 10% sodium dodecyl sulfate polyacrylamide gels and electroblotted to nitrocellulose membranes. Blots were stained with Ponceau S to confirm that protein loading was equal in all lanes. Filters were soaked in PBS to remove the Ponceau S and incubated in PBS containing 5% dried skimmed milk and 0.1% Tween 20 for 2 h to block the excess of non-specific protein-binding sites. Filters were then incubated overnight at 4°C with the primary antibodies diluted 5% dried skimmed milk and 0.1% Tween in TBS buffer, followed by secondary antibodies. Finally, the proteins were detected using an enhanced chemiluminescence western blot detection kit (Amersham).

Immunoprecipitation

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Phosphorylation of Oct-2 after Glu or 12-O-tetradecanoylphorbol-13-acetate (TPA) exposure was performed in cell monolayers treated for 2 h with 1 mm Glu or 100 nm TPA. Cell extracts were prepared and immunoprecipitated with anti-Oct-2 antibodies as follows: cells were scrapped with ice-cold PBS and, after a brief centrifugation (2500 g for 5 min), the pellet was solubilized in 200 µL of 50 mm Tris-HCl, 150 mm NaCl, 20 mm sodium orthovanadate, 20 mm sodium molybdate, 50 mm sodium fluoride, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate and 2 mm phenylmethylsulfonyl fluoride. The cell debris was removed by centrifugation (12 000 g for 5 min). The lysate was pre-absorbed with 15 µL of protein A coupled to Sepharose beads for 15 min at 4°C. The primary antibody was then incubated (10 h, 4°C) with samples of the supernatant fluid and the immune complexes were precipitated with protein A coupled to agarose beads for 2 h at 4°C. The beads were pelleted, washed several times with NET buffer (50 mm Tris HCl, 150 mm NaCl, 1 mm EDTA, 20 mm sodium orthovanadate, 20 mm sodium molibdate, 50 mm sodium fluoride, 0.25% bovine serum albumin and 0.1% NP-40, pH 7.5) and boiled for 5 min in Laemmli's sample buffer. Equal amounts of solubilized material were analyzed through 10% sodium dodecyl sulfate polyacrylamide gels and the proteins stained with Diamond phosphoprotein gel stain as indicated by the manufacturer.

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

Cell culture staining with anti-Oct-2 polyclonal antibodies was performed. The BGCs were grown on ethanol-washed and poly-l-lysine-treated (0.01 mg/mL) glass coverslips (22 × 22 mm) in the same culture conditions as described above. Cells were fixed by exposure for 10 min to methanol at −20°C, washed twice with PBS and exposed to a 1 : 200 dilution of anti-Oct-2 rabbit polyclonal antibodies or a 1 : 500 dilution of anti-KBP antisera for 60 min at room temperature. The binding of the primary antibodies was visualized using fluorescein-labeled goat anti-rabbit antibodies. Control immunolabeling was performed with the same staining procedure but using the visualizing reagents in the absence of the primary antibodies. The coverslips were mounted with Vectashield and the fluorescence examined using a confocal microscope (MRC-600; BIO-RAD, Hercules, CA, USA) with krypton argon laser.

For tissue sections, 18-day-old chick embryos were used. The cerebellum was removed immediately after killing, cryoprotected successively in 10, 20 and 30% sucrose in PBS and then sectioned coronally at 20 µm with a cryostat (Micron, Fisher Scientific, Itasca, IL, USA). Serial sections were collected in PBS and washed. The tissue was exposed for 10 min to 0.5% hydrogen peroxide in order to eliminate endogenous peroxidase activity. Non-specific tissue antibody reactions were blocked by placing the sections in 3% normal horse serum for 1 h at room temperature. Sections were then incubated for 24 h at 4°C with or without the primary antibodies. The tissue was then placed in biotinylated goat anti-rabbit serum for 1 h and, after several washes with PBS, it was incubated with avidin-biotin-horseradish peroxidase complex. Immunoreactivity was revealed with a solution of 0.05% diaminobenzidine in the presence of 10 mg/mL nickel sulfate, 10 mg/mL cobalt chloride and 0.01% hydrogen peroxide, which produce the characteristic black–purple precipitate. After 10 min the tissue was transferred to PBS to stop the reaction. Sections were mounted onto gelatin-subbed slides, dehydrated and cleared in Hemo-De (Fisher Scientific, Itasca, IL, USA) and then coverslipped with Permount (Biomedia, Foster City, CA, USA). The slides were examined under bright field on a BX41 microscope (Olympus, Melville, NY, USA) at ×4 and ×100 magnification.

Expression of Oct-2 in chick cerebellar Bergmann glia cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

The characterization of the chkbp gene and its transcriptional activation after Glu treatment of BGCs revealed the presence of a putative negative regulatory element located in the −250/−170 region (Aguirre et al. 2000, 2002). Computational analysis revealed several transcription factor-binding sites so, in order to analyze them, the negative regulatory element was sliced into three fragments, SIL-1, SIL-2 and SIL-3 (Fig. 1a). Taking into consideration that Glu induces an increase in the DNA-binding activity of activator protein 1, signal transduction activation protein 3 and cAMP response element-binding protein in BGCs (Sanchez and Ortega 1994; Aguilera and Ortega 1999; Aguirre et al. 2000, 2002), we first sought any DNA-binding activity present in nuclear extracts of Glu-stimulated cells that would bind to [32P]-labeled SIL-1, SIL-2 or SIL-3. Under standard gelshift conditions, no clear DNA–protein complexes were detected with SIL-1 or SIL-3 (not shown), whereas a defined retarded band was obtained with SIL-2 (Fig. 1b). Note that, in nuclear extracts prepared from chick fibroblast cell line CEC-32, similar protein–DNA complexes were retarded. As an octamer sequence is present in SIL-2, we decided to [32P]-label a bona fide consensus Oct-1 probe, which has been shown to bind several members of the Oct family (Dent and Latchman 1991). As depicted in Fig. 1(b), in Glu-stimulated BGC nuclear extracts, both Oct-1 and Oct-2 complexes were again detected; similar complexes were detected in CEC-32 chick fibroblasts.

image

Figure 1. The chkbp silencer is recognized by the Oct-2 transcription factor present in Bergmann glial cells. (a)  The proposed silencer region located in the chkbp gene promoter showing the three designed oligonucleotides for this region. Computational analysis was performed using the Transfact data base ( Wingender et al. 2000 ). (b)  Nuclear extracts from control [non-stimulated cells (N/S)] or glutamate (Glu)-treated Bergmann glial cells (BGCs) (1 m m for 2 h) and chick fibroblasts [CEC-32 cell line, denoted as chicken fibroblasts (FIB)] were obtained and tested by electrophoretic mobility shift assays (EMSA) using the [ 32 P] SIL-2 probe or the Oct-1 oligonucleotide containing a typical octamer consensus binding site. In (c) , EMSA was performed using the same end-labeled probes and competition assays were performed with a 100-fold excess of indicated non-labeled oligonucleotides for [ 32 P]-labeled SIL-2 and for Oct-1 competed with a 65-fold excess. –, No competitor added; OctM, Oct-1 mutated version. (d)  The presence of Oct-1 and Oct-2 was tested by western blot using anti-Oct-1 and anti-Oct-2 antibodies using nuclear extracts from BGCs. Nuclear extracts obtained from HeLa and HL60 cell lines were used as controls. (e)  Supershift assays were performed using the same anti-Oct-1 and anti-Oct-2 antibodies to detect the interaction of both factors with SIL-2 and Oct-1 and BGC nuclear extracts. Anti-p53 antibodies were used for control. Oct-1 and HeLa nuclear extracts were used to show the Oct-2 supershifted band with this specific probe. (f)  BGCs were cultured over coverslips and treated with 1 m m Glu as indicated. Immunofluorescence assay was performed with anti-Oct-2 antibody and a secondary FITC anti-rabbit. Images were obtained by confocal microscopy. Representative results of at least three independent experiments are shown.

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Using a competition approach, shown in Fig. 1(c), we were able to identify the retarded band [SIL-2 complex (sil2cx)] as an Oct-related complex. A 100-fold excess of unlabeled Oct-1 probe prevents the appearance of sil2cx and vice versa, an excess of unlabeled SIL-2 abolishes the formation of the Oct complexes. These results prompted us to demonstrate Oct-1 and Oct-2 expression in BGCs. Western blot analysis of control and Glu-stimulated BGC nuclear extracts with anti-Oct-1 or anti-Oct-2 antibodies revealed the presence of both proteins (Fig. 1d, western blot). The identity of the Oct member present in nuclear extracts that binds to SIL-2 was established via supershift experiments with anti-Oct-1 or anti-Oct-2 polyclonal antibodies. The results are shown in Fig. 1(e); only anti-Oct-2 antibodies are able to modify the migration of sil2cx, clearly suggesting that Oct-2 is present in this complex. Immunocytochemical analysis of the cultured cells not only reinforced the demonstration of the expression of Oct-2 in cultured BGCs but also showed that Glu treatment induces the nuclear translocation of Oct-2 (Fig. 1f).

Oct-2 is expressed in Bergmann glia in vivo

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

The data presented above, although leaving no doubt about the expression of Oct-2 in cultured BGCs, do not necessarily represent the in vivo situation. For this purpose, we examined paraffin-embedded serial sections of 18-day-old chick cerebellum with anti-Oct-2 polyclonal antibodies as well as anti-KBP antiserum, an established marker of Bergmann glia (Somogyi et al. 1990). As depicted in Fig. 2, Oct-2, as well as KBP, is present in the molecular layer of the cerebellar cortex. At higher magnification it is possible to observe that Oct-2 immunoreactivity is confined to the cell bodies of Bergmann glia whereas anti-KBP immunoreactivity is present in both the cell body and the extensions of these cells as previously described (Somogyi et al. 1990).

image

Figure 2. Oct-2 transcription factor is expressed in chick cerebellar Bergmann glia. Eighteen day-old chick embryos were used and tissue was prepared as in Experimental procedures. Photomicrographs of sections immunostained for Oct-2 and kainate-binding protein (KBP). Arrows indicate Bergmann glia. Magnification is indicated.

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α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

As stated above, sil2cx increases when BGCs are stimulated with 1 mm Glu; we, therefore, decided to characterize this effect. A time course was established, cells were treated with 1 mm Glu for different time periods and, as shown in Fig. 3(a), a significant augmentation in Oct-2 DNA binding was detected after a 2-h stimulatory period and continued to increase for up to 16 h. Accordingly, western blot analysis of nuclear extracts prepared from Glu-treated cells showed an increased expression of Oct-2 after a 90-min treatment with Glu. The dose dependence of the effect was explored in cells stimulated for 2 h and an EC50 value of 164 µm was determined, suggesting the involvement of iGluRs (Fig. 3b).

image

Figure 3. SIL-2 complex (sil2cx) formation is time and dose dependent and elicited through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. (a)  Nuclear extracts were obtained from Bergmann glial cells (BGCs) treated with glutamate (Glu) (1 m m ) for the indicated times and harvested immediately. EMSA was performed using [ 32 P] end-labeled SIL-2 probe and western blot (right) was performed as in Fig. 1 using anti-Oct-2 antibody. A graphic comparison of DNA-binding activity between control [non-stimulated cells (N/S)] and Glu-stimulated cells is shown. (b)  Dose–response curve of sil2cx induction where BGCs were exposed for 2 h to indicated Glu concentrations. (c)  Pharmacological profile of sil2cx induction in BGCs . BGC cultures were exposed for 2 h to Glu (1 m m ), quisqualate (Quis) (25 µ m ), 1-amino-4,5-cyclopentane- trans -1,3-dicarboxylate ( trans -ACPD) (200 µ m ), l -2-amine-4-phosphonopionic acid ( l -AP4) (200 µ m ), NMDA (400 µ m ), AMPA (400 µ m ) and kainate (KA) (1 m m ). Glu antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 µ m ) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) (25 µ m ) were used 30 min before the Glu addition. All graphs show mean values ± SE plotted from three independent experiments. *No significant difference between N/S cells and those treated with Glu and AMPA receptor antagonists DNQX or CNQX (Student's t -test).

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A pharmacological profile of the receptor involved was then obtained by exposing the cells to different agonists. Exposure of cultures to quisqualate as well as AMPA and kainic acid led to a similar increase in DNA-binding activity as in treatment with Glu (Fig. 3c), suggesting that AMPA receptors participate in the phenomena. This was confirmed when cells were exposed to the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione prior to Glu treatment (Fig. 3c).

Furthermore, as it has been established that AMPA receptors are permeable to Ca2+ ions in BGCs, it was important to evaluate whether the Glu effect would be modified in the absence of external Ca2+. The results are shown in Fig. 4(a); when the cells are treated with Glu in a Ca2+-free medium, the intensity of the sil2cx complex is almost the same as that of non-stimulated cells. As expected, the binding of stimulating protein 1 to its consensus oligonucleotide is not affected by Glu (Fig. 4a, right). In line with a Glu-dependent Oct-2 nuclear translocation, western blot analysis of these nuclear extracts showed a diminished Oct-2 immunoreactivity when the cells were treated with Glu in the absence of external Ca2+ (Fig. 4b).

image

Figure 4. SIL-2 complex (sil2cx) induction by glutamate (Glu) is dependent on Ca 2+ influx together with protein kinase C (PKC) and phosphatidyl inositol 3-kinase (PI-3K) activation. (a) EMSA using the SIL-2 probe was performed with NE from control or Glu-treated Bergmann glial cells (BGCs) in Ca2+-free medium. The same NE were assayed for stimulating protein 1 (Sp-1) DNA binding. (b)  Western blot analysis of NE with anti-Oct-2 antibodies. (c)  Nuclear extracts (NE) were obtained from BGCs stimulated with Glu (1 m m , 2 h) or with the PKC activator, phorbol ester 12- O -tetradecanoylphorbol-13-acetate (TPA, 100 n m ). α[ N -(2-Aminoethyl)-5-isoquinolinesulfonamide, 2HCl] (H-9) was used as PKC inhibitor and wortmannin (Wor) (100 n m ) for PI-3K inhibition; both were added 30 min before stimulation. A representative experiment of at least three independent experiments is shown. N/S, non-stimulated cells.

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The activation of PKC by Glu receptors has been demonstrated in a number of systems, including BGCs (Cid and Ortega 1993; Aguirre et al. 2000). Therefore, we decided to explore if the increase in Oct-2 DNA binding elicited by AMPA receptors involved PKC activation. For this purpose, we stimulated BGCs in the presence of α[N-(2-aminoethyl)-5-isoquinolinesulfonamide, 2HCl] (H-9), a broad-spectrum serine/threonine protein kinase blocker that is PKC specific when used at a concentration of 80 µm, and a PKC activator, the phorbol ester TPA. As expected, 100 nm TPA is sufficient to mimic the Glu effect and, conversely, pre-exposure of BGCs to H-9 reduces the intensity of the sil2cx complex (Fig. 4c). We have recently shown that phosphatidyl inositol 3-kinase is involved in AMPA receptor signaling (Aguirre et al. 2000; Gamboa and Ortega 2002). In this respect, we explored the possibility that wortmannin, a phosphatidyl inositol 3-kinase blocker, could reverse the Glu-stimulated appearance of sil2cx. As shown in Fig. 4(c), this is indeed the case; pre-exposure of BGCs to 100 nm wortmannin reverses the Glu effect.

Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

The phosphorylation status of Oct-2 has been shown to be important for the DNA-binding capacity of this transcription factor. In fact, its phosphorylation pattern dictates whether it binds to a certain promoter region (Wirth et al. 1991; Lillycrop and Latchman 1992; Grenfell et al. 1996; Liu et al. 1996; Pevzner et al. 2000). In order to gain an insight into the Oct-2 phosphorylation status under our experimental conditions, cell extracts were prepared from control, Glu- and TPA-stimulated cells and immunoprecipitated with anti-Oct-2 antisera. The immune complexes were analyzed in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis slab gels and stained with the novel Diamond phosphoprotein gel stain. As depicted in Fig. 5, both Glu and TPA treatment resulted in Oct-2 phosphorylation. This increase in Oct-2 phosphorylation is time dependent. These results favor the notion that Oct-2 has to be phosphorylated in order to bind SIL-2.

image

Figure 5. Glutamate (Glu) regulates Oct-2 at the transcriptional and post-translational levels. (a) Phosphorylation of Oct-2 after Glu or 12- O -tetradecanoylphorbol-13-acetate (TPA) exposure was tested when cell monolayers were treated for 2 h or indicated times with 1 m m Glu or 100 n m TPA for 2 h; cell extracts were prepared and immunoprecipitated with anti-Oct-2 antibodies. The immune complexes were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the gels stained with Diamond phosphoprotein gel stain (Molecular Probes). (b)  Glu-dependent SIL-2 complex (sil2cx) formation precedes Oct-1 and Oct-2 increased protein levels. Cells were treated for the indicated time periods and returned to normal media for up to 4 h. The cells were collected and nuclear extracts prepared for western blot analysis with anti-Oct-1 or anti-Oct-2 antibodies and for EMSA with the SIL-2 probe. (c)  The Glu-dependent increase in SIL-2 DNA binding is prevented by the transcriptional inhibitor actinomycin-D (AD) or the translational inhibitor cycloheximide (CHX). Cells were treated with 1 m m Glu for 4 h in the presence or absence of AD or CHX, nuclear extracts were prepared and analyzed by EMSA with the SIL-2 probe, stimulating protein 1 (Sp-1) probe or tested by western blot using anti-Oct-2 antibody (d).  Representative results are shown from three experiments with similar results. BGC, Bergmann glial cell; N/S, non-stimulated cells .

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The rather slow kinetics of the induction of sil2cx in Glu-treated BGCs (Fig. 3), together with the increase in Oct-2 protein levels (Fig. 3), suggests that the signaling events triggered through AMPA receptors are not restricted to a change in the phosphorylation status of Oct-2. The DNA-binding activity persists up to 16 h after Glu exposure, suggesting that a long-term effect might be taking place. In order to clarify this issue, we first decided to explore the minimum exposure time to Glu needed to induce an increase in both the sil2cx formation and Oct-1 and Oct-2 immunoreactivity. Confluent BGC cultures were exposed to Glu for 5, 15, 30 or 60 min, the culture media was replaced with fresh medium without the agonist and the cells were collected 2 h after the initial stimulation. Under such conditions it is quite evident that sil2cx is formed before any substantial change in Oct-2 protein levels are detected by western blot (Fig. 5b). These results support the hypothesis that AMPA receptor activation regulates Oct-2 DNA binding at two levels, phosphorylation and possibly by transcriptional activation.

To this end, we exposed BGCs to Glu for 1 h in the presence of the transcription inhibitor actinomycin-D or the translation inhibitor cycloheximide. The cells were collected after 3 h and nuclear extracts were prepared and analyzed both by EMSA with SIL-2 and by western blots with anti-Oct-2 antibodies. The results are shown in Fig. 5(c); both inhibitors block the Glu-mediated formation of sil2cx, whereas a less evident effect of cycloheximide or actinomycin-D is present at the level of the immunoreactive protein (Fig. 5d), again reinforcing the idea of two levels of regulation. As a control, we performed stimulating protein 1 DNA binding with the same nuclear extracts and, as expected, no changes were found in control versus treated cells (Fig. 5c).

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References

The BGCs in the cerebellum elaborately ensheath Purkinje cell dendrites and synapses. They express Ca2+-permeable iGluRs essential for maintaining both structural and functional connection between the glial cell and the glutamatergic synapses (Iino et al. 2001; Watanabe 2002). Adenovirally-mediated conversion of these receptors to Na+-permeable receptors retracts the glial processes that grap the synapses on Purkinje cell dendritic spines and retard removal of synaptically released Glu. Clearly, Glu-induced changes in gene expression are implicated in these processes (Iino et al. 2001).

In astrocytes, Glu regulates several transcription factors; among them activator protein 1 and nuclear factor kappa B are important for gene expression regulation (Condorelli et al. 1993; O'Neill and Kaltschmidt 1997). In BGCs, Glu triggers the binding to DNA of transcription factors, such as activator protein 1, signal transduction activation protein 3 and cAMP response element-binding protein (Sanchez and Ortega 1994; Aguilera and Ortega 1999; Aguirre et al. 2000, 2002), and also regulates KBP expression (Aguirre et al. 2000, 2002). The fact that Glu receptor activation is involved in a wide variety of learning paradigms and that KBP is up-regulated in an imprinting stimulus strongly suggest that glial Glu receptors might play a role in neuronal plasticity (Aguirre et al. 2000).

In the present work, we show that BGC AMPA receptors regulate Oct-2 binding activity as well as its protein levels. These findings were unexpected in view of the reported rather restricted expression of this factor. For instance, within the nervous system, Oct-2 has only been found in neuronal cells (Lillycrop et al. 1991). A plausible explanation for our results is the nature of the cultured cells. While most authors use cultured astrocytes prepared from whole brain or cerebral cortex astrocytes, we cultured cerebellar radial glia, which is the early glia in development and does not undergo the so-called ‘astrocytic conversion’ persisting in adulthood (Petryniak et al. 1990). It should be kept in mind that radial glia is neurogenic not only during development but also in adulthood (Petryniak et al. 1990). It is, therefore, plausible that radial glia cells express genes which are regarded as neuronal specific, such as the NMDA receptor NR1 subunit (Lopez et al. 1997).

Oct-2 is an ambivalent transcription factor, presenting multiple isoforms generated through alternative splicing that are expressed in a tissue-specific manner. The different Oct-2 isoforms, with molecular weights ranging from 49 to 75 kDa, have a characteristic transcriptional repressor activity in neuronal cells (Thomas et al. 1998; Latchman 1999). The fact that we were able to identify Oct-2 as a component of a protein complex that binds to a putative negative regulatory element in a glial-specific gene further supports the role of Oct-2 as a repressor in the CNS (Fig. 1). One could argue that Oct-2 expression in cultured BGCs could certainly be misleading and far from the in vivo situation. The results presented in Fig. 2 clearly demonstrate Oct-2 expression in the cerebellar molecular layer. This immunoreactivity matches that of KBP, establishing the identity of these cells as Bergmann glia (Somogyi et al. 1990).

The Glu-mediated increase in Oct-2 nuclear localization and DNA binding (Fig. 1) is a receptor-mediated effect with a pharmacological profile corresponding to AMPA receptors (Fig. 3). The possibility that metabotropic Glu receptors are involved in the Glu response, in view of the fact that quisqualate is capable of inducing a significant increase in Oct-2 binding, is discarded as the Glu effect is not mimicked by 1-amino-4,5-cyclopentane-trans-1,3-dicarboxylate even at a 1-mm concentration and the effect being sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione settles the point (Fig. 3). As expected, removal of external Ca2+ reduces Oct-2–sil2cx formation without affecting the binding of other transcriptional factors such as stimulating protein 1, again strongly suggesting the involvement of Ca2+-permeable AMPA receptors. Regarding the time course of the Glu effect, it is clear that a sustained augmentation of Oct-2 DNA binding is obtained. Such a prolonged increase in binding (up to 16 h) is indicative of a possible up-regulation of the oct-2 gene (Fig. 3). The use of cycloheximide and actinomycin-D is sufficient to completely block the Glu effect, both at the level of the DNA binding as well as the increase in the nuclear Oct-2 levels (Fig. 5). These results prompt us to speculate that Glu, acting through Ca2+-permeable AMPA receptors, on the one hand leads to Oct-2 phosphorylation and, therefore, augments Oct-2 DNA binding while, on the other hand, it might activate oct-2 transcription (Fig. 5).

In summary, we demonstrate here the expression of Oct-2 in Bergmann glia. We also provide evidence for an AMPA receptor-mediated regulation of its ability to bind DNA. Work currently under way in our laboratory is aimed at establishing the signal transduction pathways involved in oct-2 gene expression regulation by glial Glu receptor activation.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and stimulation protocol
  6. Electrophoretic mobility shift assays
  7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots
  8. Immunoprecipitation
  9. Immunohistochemistry
  10. Results
  11. Expression of Oct-2 in chick cerebellar Bergmann glia cells
  12. Oct-2 is expressed in Bergmann glia in vivo
  13. α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor stimulation increases Oct-2 DNA-binding activity
  14. Glutamate exposure regulates Oct-2 at the transcriptional and post-translational levels
  15. Discussion
  16. Acknowledgements
  17. References
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