Glutamate Regulates Kainate-binding Protein Expression in Cultured Chick Bergmann Glia through an Activator Protein-1 Binding Site

The expression of the chick kainate-binding protein, a member of the ionotropic glutamate receptor family, is restricted to the cerebellum, specifically to Bergmann glia. Glutamate induces a membrane to nuclei signaling involved in gene expression regulation. Exposure of cultured chick Bergmann glia cells to glutamate leads to an increase in kainate binding protein and mRNA levels, suggesting a transcriptional level of regulation. The 5′ proximal region of the chick kainate binding gene was cloned and transfected 4into Bergmann glia cells. Three main regulatory regions could be defined, a minimal promoter region, a negative regulatory region, and interestingly, a glutamate-responsive element. Deletion of this element abolishes the agonist effect. Moreover, electrophoretic mobility shift assays, cotransfection experiments, and site-directed mutagenesis clearly suggest that the glutamate effect is mediated through an AP-1 site by a Fos/Jun heterodimer. The present results favor the notion of a functional role of kainate-binding protein in glutamatergic cerebellar neurotransmission. The expression of the chick kainate-binding protein, a member of the ionotropic glutamate receptor family, is restricted to the cerebellum, specifically to Bergmann glia. Glutamate induces a membrane to nuclei signaling involved in gene expression regulation. Exposure of cultured chick Bergmann glia cells to glutamate leads to an increase in kainate binding protein and mRNA levels, suggesting a transcriptional level of regulation. The 5′ proximal region of the chick kainate binding gene was cloned and transfected 4into Bergmann glia cells. Three main regulatory regions could be defined, a minimal promoter region, a negative regulatory region, and interestingly, a glutamate-responsive element. Deletion of this element abolishes the agonist effect. Moreover, electrophoretic mobility shift assays, cotransfection experiments, and site-directed mutagenesis clearly suggest that the glutamate effect is mediated through an AP-1 site by a Fos/Jun heterodimer. The present results favor the notion of a functional role of kainate-binding protein in glutamatergic cerebellar neurotransmission. kainate-binding protein glutamate-responsive element α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid kainic acid Bergmann glia cells activator protein 1 phosphate-buffered saline polymerase chain reaction reverse transcriptase-PCR chloramphenicol acetyltransferase base pair(s) Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. To elicit its functions, Glu activates two types of receptors: ligand-gated ion channels and metabotropic receptors coupled to G-proteins (1Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3689) Google Scholar). Kainate-binding proteins (KBPs)1 are members of the ionotropic Glu receptors' gene family, the expression of these proteins is enriched in non-mammalian vertebrates (2Henley J.M. Trends Pharmacol. Sci. 1994; 15: 182-190Abstract Full Text PDF PubMed Scopus (42) Google Scholar). In the chick, KBP is expressed as a 49-kDa glycosylated polypeptide that is restricted to the cerebellum. In situ hybridization and immunohistochemical studies have shown that KBP is expressed exclusively in Bergmann glia (3Gregor P. Eshhar N. Ortega A. Teichberg V.I. EMBO J. 1988; 7: 2673-2679Crossref PubMed Scopus (63) Google Scholar, 4Somogyi P. Eshhar N. Teichberg V.I. Roberts D.B. Neuroscience. 1990; 35: 9-30Crossref PubMed Scopus (103) Google Scholar). The chick KBP is the best characterized of the non-mammalian vertebrate KBPs and is the major binding site for kainate within the cerebellum. Several lines of evidence suggest a functional role of chick KBP; for example, the expression of KBP follows by several days the onset of Bergmann glia development and matches the time of migration of granular cells (2Henley J.M. Trends Pharmacol. Sci. 1994; 15: 182-190Abstract Full Text PDF PubMed Scopus (42) Google Scholar). In addition, KBP expression is up-regulated by an imprinting stimulus in ducks (5Kimura N. Kurosawa N. Kodo K. Tsukada Y. Brain. Res. Mol. Brain. Res. 1993; 17: 351-355Crossref PubMed Scopus (13) Google Scholar). Nevertheless, with the exception of toad KBP, no functional properties have been reported for this protein. No ligand-gated ion channel activity is observed, even when chick KBP is coexpressed with other ionotropic receptor subunits. Interestingly, when the transmembranal domain of the chick KBP is linked to the Glu binding site of membrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) receptors in a chimera construction, this transmembranal domain can function as an ionic pore (6Villmann C. Bull L. Hollmann M. J. Neurosci. 1997; 17: 7634-7643Crossref PubMed Google Scholar). Bergmann glia extends processes through the molecular layer, ensheathing Purkinje cell dendrites and the excitatory synapses between Purkinje cells and the parallel and climbing fibers. Because of this specific localization, it has been suggested that these cells participate in the modulation of the efficacy of excitatory synapses surrounded by them (4Somogyi P. Eshhar N. Teichberg V.I. Roberts D.B. Neuroscience. 1990; 35: 9-30Crossref PubMed Scopus (103) Google Scholar). Bergmann glia cells (BGC) carry in their membranes AMPA/KA, N-methyl-d-aspartate, and metabotropic receptors (7López T. López-Colomé A.M. Ortega A. FEBS Lett. 1997; 405: 245-248Crossref PubMed Scopus (60) Google Scholar). The activation of these receptors promotes calcium influx, phosphoinositide hydrolysis, PKC translocation to the membrane, activation of the MAPK (mitogen-activated protein kinase) pathway, increase in DNA binding activity of the activator protein-1 (AP-1) and changes in Glu receptors expression (8López T. López-Colomé A.M. Ortega A. Brain. Res. Mol. Brain. Res. 1998; 58: 40-46Crossref PubMed Scopus (29) Google Scholar). Parallel fiber stimulation induces depolarization of Bergmann glia, an effect mediated by AMPA/KA receptors and by the Na+-dependent Glu transporter (9Clark B.A. Barbour B. J. Physiol. 1997; 502: 335-350Crossref PubMed Scopus (160) Google Scholar). These responses suggest that long term changes within Bergmann glial cells occur as a consequence of synaptic activity. In this sense, chick KBP could be an important molecule related to long term changes in glial function, glia-neuron communication, or synaptic plasticity. The relative expression of the various Glu receptors is not static but is remodeled during development, isquemia, seizures, repetitive activation of afferents, or chronic administration of a variety of drugs (10Myers S.J. Dinglidine R. Borges K. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 221-241Crossref PubMed Scopus (94) Google Scholar). Nevertheless, little is known of the molecular identity of the promoter regions involved in such regulation, mainly because in the promoter regions reported thus far, none of them predicts the interaction of inducible transcription factors. An interesting exception is the chkbp promoter, in which a putative AP-1 binding site has been reported (11Gregor P. Yang X. Mano I. Takemura M. Teichberg V.I. Brain. Res. Mol. Brain. Res. 1992; 16: 179-186Crossref PubMed Scopus (15) Google Scholar, 12Eshhar N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google Scholar). In the present work, we provide evidence for a Glu-induced transcriptional regulation of KBP expression mediated through an AP-1 site. Chick cerebellar BGC were prepared as detailed elsewhere (8López T. López-Colomé A.M. Ortega A. Brain. Res. Mol. Brain. Res. 1998; 58: 40-46Crossref PubMed Scopus (29) Google Scholar). Briefly, 14-day-old chick embryos were used, and the cerebellum was dissected and homogenized mechanically. Cells were plated at a density of 1 × 106 ml−1 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 50 μg/ml gentamicin. The cells were incubated at 37 °C in 5% CO2 and used after 5–6 days in culture. Confluent monolayers were exposed to the indicated concentrations of agonists for varying periods of time; antagonists were added 10 min before the agonists. Incubation was stopped by removing the medium, and samples were processed as detailed below. The cells were harvested and washed several times with 10 mmK2HPO4/KH2PO4, 150 mm NaCl, pH 7.4 (PBS). 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), and 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 50 μg) were resolved in 10% SDS-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 1 h to block the excess of nonspecific protein binding sites. Filters were then incubated for 12 h at 4 °C with primary antibodies diluted in 0.25% bovine serum albumin, 0.1% Tween 20 in PBS buffer followed by secondary antibodies. The antibodies used were: polyclonal anti-KBP, polyclonal anti-c-Jun, polyclonal anti-p53 (Santa Cruz Biotechnology), and anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Finally, the proteins were detected using an ECL chemiluminescence kit (Amersham Pharmacia Biotech). For semiquantitative RT-PCR, reverse-transcription was performed as described previously (7López T. López-Colomé A.M. Ortega A. FEBS Lett. 1997; 405: 245-248Crossref PubMed Scopus (60) Google Scholar). Total RNA was isolated from confluent monolayers as described by Chomczynski and Sacchi (13Chomczynski P. Sacchi M. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65695) Google Scholar). First-strand cDNA was synthesized using 2 μg of total RNA, 200 units of Moloney murine leukemia virus-reverse transcriptase, and 40 pmol of oligo(dt) as primer. The coamplification of an internal control housekeeping S17 chick ribosomal protein mRNA was performed. Cycling conditions were 95 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min, by 24 cycles. Equal aliquots of each PCR reaction were removed and analyzed by 1.2% agarose-gel electrophoresis. Data were quantified by scanning the labeled bands, and the optical densities of KBP were normalized to the S17 signal. The oligonucleotides used as primers were: KBP sense, 5′-GCGAATTCGTGGGAGATGGGAAGTATGGC-3′; KBP antisense,: 5′-GCGGTACCTATGGTGAAGAGCCACCA-3′; S17 sense, 5′-TACACCCGTCTGGGAACGAC-3′; S17 antisense, 5′-CCGCTGGATGCGCTTCATCAG-3′. Linear amplification was found between 24 and 30 cycles as determined by scanning of the ethidium bromide staining of the electrophoresed PCR products (data not shown). The pKS-KBPNet plasmid that contains the entire 1.7 kilobase pairs of chKBP cDNA (a gift of Prof. V. I. Teichberg, Weizman Institute) was labeled by the random primer method and used as probe. Approximately 15 μg of total RNA from cultured BGC were electrophoresed, transferred by capillarity to nitrocelluose membranes, hybridized for 16 h at 42 °C, and exposed to x-ray films. The specific amount of KBP mRNA was normalized against the quantification of RNA on Nothern membranes using ethidium bromide staining and optical scanning as described by Eykholtet al. (14Eykholt R.L. Mitchell M.D. Marvin K.W. BioTechniques. 2000; 28: 868-870Crossref Scopus (8) Google Scholar). All plasmids were constructed and sequenced using standard techniques. The plasmid p435kbpCAT contains the entire chick KBP 5′-noncoding region cloned in the pCAT-Basic vector (Promega) amplified by PCR using the primers kbpup (5′-ACATGCATGCAAAGGCCTTTCTTTCAGC-3′) and kbplow (3′-CTAGTCTAGAAAACTTCAGCAATTCCTTCA-5′) and chick cerebellum DNA. Serial deletions of p435kbpCAT were made by cuts in the appropriated sites to generate p250kbpCAT (HindIII) and p170kbpCAT (BspMI) constructs. For AP-1 site mutagenesis, three point mutations were introduced in the context of p435kbpCAT (p435kbpAp1M), using a QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following oligonucleotides were used to generate mutations in AP-1 control element: 5′-CCCCAACTGATCTGACttcATCACGGAG-3′; 5′-CTTTCTCCGTGATgaaGTCAGATCAGTTGGGG-3′. The lowercase letters indicate the mutated bases. PCR was performed in a 50-μl volume withPfu polymerase (Stratagene), 5 ng of DNA template and 125 ng of each oligonucleotide using the manufacturer's conditions. The PCR product was treated with DpnI (10 units) for 60 min at 37 °C and DNA-transformed in Escherichia coli DH5α. Plasmids were prepared from individual colonies and sequenced to confirm the introduced mutations. The double-stranded wild type (see below) and mutant as used for oligonucleotides were cloned in site of the promoter and of the vector (Promega) that contains in the plasmids and The wild type oligonucleotide was cloned in the p250kbpCAT All were for the and sequenced to wild type or mutated Pharmacia Biotech). of the expression plasmids and used in cotransfection and were in BGC using with μg of with glutamatergic was performed 24 h for the indicated time periods and were as Cells were harvested in buffer mm Tris-HCl pH 1 mm 15 mm lysed with three cycles in pH and at for Equal amount of protein μg) were incubated with of Pharmacia and at 37 were by and quantified using an were expressed as the for the activity in the pCAT-Basic vector and are expressed as relative to control cell were the using the indicated amounts of the expression were prepared as described previously A. A. P. J. Full Text Full Text PDF PubMed Scopus Google Scholar). All of the protease inhibitors to concentration was by the method Anal. Biochem. PubMed Scopus Google Scholar). (approximately 15 μg) from control or BGC 2 for indicated were incubated on with 1 μg of as nonspecific (Amersham Pharmacia and 1 ng of double-stranded oligonucleotides as The reaction were incubated for 10 min at 4 °C and electrophoresed in or gels using a ionic buffer. The gels were dried and exposed to an For the reaction were with the indicated amount of oligonucleotide before the of labeled DNA. For experiments, with the were incubated at 4 °C with Cruz or anti-p53 Cruz for 2 h to electrophoresis. of cultured chick BGC with 1 mm Glu a of the 49-kDa KBP polypeptide as detected with an against the protein 1 The is after 12 h of Glu and is present after 24 h of agonist The of the effect was and an of 200 was determined 1 of the receptors involved in chick KBP was Exposure of the for 12 h to a concentration of kainate as as to 200 10 or to a increase in KBP as in with 1 mm Glu 2 suggesting the of ionotropic and metabotropic receptors in the was when specific antagonists were used to block the Glu effect. with the specific antagonists at a concentration of 50 at 5 acid at in a in the increase in of KBP cells were incubated for 12 h with the indicated Glu 1 200 10 or the antagonists 50 5 acid were added 30 min before KBP were quantified as in are the of three To the level of regulation modified to Glu we chick KBP mRNA by of semiquantitative from control and using the ribosomal protein as an internal control 1993; PubMed Google Scholar). Linear amplification was found between 24 and 30 as by scanning of the ethidium bromide staining of the electrophoresed PCR products shown). of the cells with 1 mm Glu in a increase in chKBP mRNA These results were by the amount of KBP in cells from a transcriptional regulation of the gene by Glu receptor activation in The previously reported as the putative promoter of chick KBP not been characterized (11Gregor P. Yang X. Mano I. Takemura M. Teichberg V.I. Brain. Res. Mol. Brain. Res. 1992; 16: 179-186Crossref PubMed Scopus (15) Google Scholar, 12Eshhar N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google Scholar). to the transcriptional regulation of KBP, we isolated chick DNA and performed PCR with specific primers to the putative promoter N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google Scholar). The of was cloned a plasmid and in the changes in with the previously reported (11Gregor P. Yang X. Mano I. Takemura M. Teichberg V.I. Brain. Res. Mol. Brain. Res. 1992; 16: 179-186Crossref PubMed Scopus (15) Google Scholar, 12Eshhar N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google Scholar). To functional assays, the putative promoter was the plasmid and BGC were transfected in conditions not with The p435kbpCAT to a expression of the gene in with p250kbpCAT or p170kbpCAT 5 Deletion of or of the 5′ region the promoter activity after suggesting that the region could several negative regulatory transcriptional activity in conditions can be in the with 1 mm Glu of cultured BGC transfected with the in a increase in activity 5 Exposure to 1 mm this effect 5 Interestingly, when the p250kbpCAT and p170kbpCAT were for Glu none of them to the agonist 5 These results clearly suggest that the region the to Glu a element The promoter of chkbp several putative binding sites for inducible transcription P. Yang X. Mano I. Takemura M. Teichberg V.I. Brain. Res. Mol. Brain. Res. 1992; 16: 179-186Crossref PubMed Scopus (15) Google and 12Eshhar N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google Scholar). that Glu promotes an increase in the binding activity of AP-1 in Bergmann glial cells Ortega A. 1994; PubMed Scopus Google it was to that Glu be KBP expression through the putative AP-1 binding within its Interestingly, an is present at the to and of with the using electrophoretic mobility shift as as functional with several constructs. containing the putative chick AP-1 site was labeled and incubated with prepared from control or in induces an increase in the of two as as the of a a increase in binding to the AP-1 site is by (8López T. López-Colomé A.M. Ortega A. Brain. Res. Mol. Brain. Res. 1998; 58: 40-46Crossref PubMed Scopus (29) Google Scholar, Ortega A. 1994; PubMed Scopus Google Scholar). that with not in an increase on binding to the element P. Ortega A. Res. 1999; PubMed Scopus Google Scholar). The of the chick AP-1 site was performed using an double-stranded Several specific were with of activity was in BGC treated with the agonist An excess of or of abolishes the to the The concentrations of a mutated of or a oligonucleotide not for a binding an excess of is to for binding 12 thus suggesting that the AP-1 members are to the site in the with polyclonal antibodies in a of the mobility of the and a These results suggest that is of the transcription to promoter Glu the was as shown in C. was used to the role of the site in the Several changes in for AP-1 binding were introduced in the context of in of abolishes the Glu that the Glu effect. the element is of Glu-induced transcriptional activity to a promoter, the that is present in the vector activity is on the and of the mutated or not Interestingly, when the site is to the chkbp region, a transcriptional increase is to Glu In with the a of BGC from cells a increase in and when the amount of or is by of the or expression with the p435kbpCAT a increase in activity is and are in the of AP-1 binding to the chkbp stimulation of Glu has that Glu not its excitatory to but with glia cells. Because and glia the of Glu an role for glia cells in is on cultured glial Glu the of receptor activates the of and the binding to DNA of inducible transcription V. C. A. X. 1999; PubMed Google Scholar, Neurosci. Lett. 1993; PubMed Scopus Google Scholar). In Glu changes these in gene induces the expression of and the transcriptional and Y. S. K. I. Y. M. Res. Mol. Res. 1994; PubMed Scopus Google Scholar). In gene expression has been reported in for following the expression of the Glu is up-regulated PubMed Scopus Google Scholar). for protein and mRNA has been for long term J. Physiol. 1999; PubMed Scopus Google Scholar). the of regulatory of Glu-induced gene expression in glial cells is a to modulation of glial cells in or by Glu has been in cultured BGC Ortega A. 1994; PubMed Scopus Google Scholar). Moreover, an imprinting stimulus leads to an increase in Bergmann KBP mRNA in ducks (5Kimura N. Kurosawa N. Kodo K. Tsukada Y. Brain. Res. Mol. Brain. Res. 1993; 17: 351-355Crossref PubMed Scopus (13) Google Scholar). The that Glu receptor activation is involved in the of a variety of that Glu receptors a role in KBP gene expression regulation. is the Glu receptor activation results in of the effect is mediated through ionotropic as as metabotropic suggesting that the signaling by these receptors or that the level of regulation modified is each receptor to be for ionotropic signaling in cultured BGC an increase in AP-1 DNA binding Ortega A. 1994; PubMed Scopus Google Scholar). that an AP-1 site is present in the putative promoter region of chkbp N. Hunter C. Wenthold R.J. Wada K. FEBS Lett. 1992; 297: 257-262Crossref PubMed Scopus (13) Google we that the increase in KBP detected Glu was the of a transcriptional regulation through this AP-1 site. it was important to that the reported putative promoter region could the transcription of a the activity be inducible by or of this AP-1 site the Glu effect. these conditions were but three main regions could be the promoter region, containing the and with a transcriptional activity but not inducible by a region containing negative regulatory the promoter but by a region, in which the AP-1 site is and for major role for the Fos/Jun in transcription is for a of prepared from cells are to the mobility of a AP-1 the AP-1 site. Moreover, this oligonucleotide with the AP-1 containing a of the putative element for the binding of proteins of Glu exposed cells. antibodies as as antibodies the could in to an increase in AP-1 Glu the binding of other putative to within this region that the effect. Because of or results in a increase in promoter activity that the Glu with other be mutagenesis of this when was to the promoter, it was to Glu is important to that when is to the p250kbpCAT which is not to Glu 5 a increase in transcriptional activity is after Glu of this to Glu is that within the region, the binding of other transcription be the AP-1 In this be the because p250kbpCAT a increase in promoter activity as with p435kbpCAT 5 Moreover, several putative transcription binding sites have been to the region detailed of this is in in is important to that negative regulatory be in the region, because of this results in a increase in activity in p170kbpCAT with the p250kbpCAT of putative in the negative regulatory region not a known binding site. Nevertheless, binding sites are in this region, and interestingly, has been reported to as an transcription as activator or in and cells Neurosci. Lett. 1993; PubMed Scopus Google Scholar). detailed is to the interaction and functional role of each in the context of Glu-induced transcriptional activity. In the present results for the a and provide a major to present of gene expression regulation. suggest that the function of KBP it a role in long term changes in glia The the of