Neuronal Excitation-driven and AP-1-dependent Activation of Tissue Inhibitor of Metalloproteinases-1 Gene Expression in Rodent Hippocampus
1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês
10.1074/jbc.274.40.28106
ISSN1083-351X
AutoresJacek Jaworski, I W Biedermann, Joanna Łapińska, Arek Szklarczyk, Izabela Figiel, Dorota Konopka‐Postupolska, Dorota Nowicka, Robert K. Filipkowski, Michal Hetman, Anna Kowalczyk, Leszek Kaczmarek,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoUnderstanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling. Understanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling. Elevated DNA-binding activity of AP-1, 1The abbreviations used are:AP-1activatory protein 1TIMP-1tissue inhibitor of metalloproteinases-1PTZpentylenetrazoleKAkainateEMSAelectrophoretic mobility shift assayLacZEscherichia coli β-galactosidase genePBSphosphate-buffered salineDMEMDulbecco's modified Eagle's mediumGFPgreen fluorescent proteinECMextracellular matrixMMPmatrix metalloproteinaseswtwild type a dimeric transcription factor composed of Fos and Jun proteins, as well as increased expression of its components have been observed repeatedly in a variety of neuronal activation phenomena (for reviews, see Refs.1Morgan J.I. Curran T. Annu. Rev. Neurosci. 1991; 14: 421-451Crossref PubMed Scopus (2475) Google Scholar, 2Hughes P. Dragunow M. Pharmacol. Rev. 1995; 47: 133-178PubMed Google Scholar, 3Kaczmarek L. Chaudhuri A. Brain Res Rev. 1997; 23: 237-256Crossref PubMed Scopus (231) Google Scholar, 4Herdegen T. Leah J.D. Brain Res. Rev. 1998; 28: 370-490Crossref PubMed Scopus (1167) Google Scholar). The use of c-Fos labeling of neurons has proven to be especially useful in delineating functional pathways in the brain (for review, see Refs. 2Hughes P. Dragunow M. Pharmacol. Rev. 1995; 47: 133-178PubMed Google Scholar and 3Kaczmarek L. Chaudhuri A. Brain Res Rev. 1997; 23: 237-256Crossref PubMed Scopus (231) Google Scholar). Furthermore, it became clear that AP-1 could serve as a prototypical excitation-responsive transcription factor (1Morgan J.I. Curran T. Annu. Rev. Neurosci. 1991; 14: 421-451Crossref PubMed Scopus (2475) Google Scholar, 2Hughes P. Dragunow M. Pharmacol. Rev. 1995; 47: 133-178PubMed Google Scholar, 3Kaczmarek L. Chaudhuri A. Brain Res Rev. 1997; 23: 237-256Crossref PubMed Scopus (231) Google Scholar). activatory protein 1 tissue inhibitor of metalloproteinases-1 pentylenetrazole kainate electrophoretic mobility shift assay Escherichia coli β-galactosidase gene phosphate-buffered saline Dulbecco's modified Eagle's medium green fluorescent protein extracellular matrix matrix metalloproteinases wild type However, despite the accumulated knowledge about expression patterns of c-Fos, AP-1, etc., in the brain in response to various stimuli, very little is known about physiological (and possibly pathological) function(s) of this transcription factor. An obvious obstacle in elucidating this function is the lack of reliable interventive strategies to affect AP-1 within nervous tissue in vivo. An alternative approach is the identification of AP-1-driven genes. However, only very few such targets in the brain have so far been identified, and moreover, none of them apparently unequivocally (see Ref. 4Herdegen T. Leah J.D. Brain Res. Rev. 1998; 28: 370-490Crossref PubMed Scopus (1167) Google Scholar, for discussion). This situation stems from the fact that this task is very complex and requires a multitude of approaches, correlative in the brain and functional in the culture dish. Recently, several pieces of information have pointed to the genetimp-1, encoding the tissue inhibitor of metalloproteinases (TIMP-1) as a possible AP-1 target in the central nervous system. Increased expression of this gene has been reported in rat brain following treatment with kainate, a glutamate receptor agonist as well as pro-convulsive and neurotoxic drug that is also well known to activate AP-1 (1Morgan J.I. Curran T. Annu. Rev. Neurosci. 1991; 14: 421-451Crossref PubMed Scopus (2475) Google Scholar, 5Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-722Crossref PubMed Scopus (437) Google Scholar, 6Rivera S. Tremblay E. Timsit S. Canals O. Ben-Ari Y. Khrestchatisky M. J. Neurosci. 1997; 17: 4223-4235Crossref PubMed Google Scholar, 7Kaminska B. Filipkowski R.K. Biedermann I.W. Konopka D. Nowicka D. Hetman M. Dabrowski M. Gorecki D. Lukasiuk K. Szklarczyk A.W. Kaczmarek L. Acta Biochim. Pol. 1997; 44: 781-789Crossref PubMed Scopus (22) Google Scholar). At the same time, Bugno et al. (8Bugno M. Greave L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordula T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar), Logan et al. (9Logan S.K. Garabedian M.J. Campbell C.E. Werb Z. J. Biol. Chem. 1996; 271: 774-782Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) as well as Botelho et al. (10Botelho F.M. Edwards D.M. Richards C.D. J. Biol. Chem. 1998; 273: 5211-5218Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) identified three major promoter elements located in the vicinity of the transcription initiation site, including the one interacting with AP-1 transcription factor, to be of critical importance in control of inducible timp-1 expression in non-neuronal cells in vitro. The present studies were designed to test the hypothesis that AP-1 may control timp-1 gene expression in rodent hippocampus in response to enhanced neuronal excitation. Such a hypothesis appears to be very attractive, since if proven, could reveal an important biological function of AP-1. Wistar male rats weighing 250–300 g from the Nencki Institute animal facility were used for the studies. In the experiments with animals, the rules established by the Ethical Committee on Animals Research of the Nencki Institute and based on national laws were strictly followed. Intraperitoneal administration of either kainate (10 mg/kg) or pentylenetetrazole (PTZ, 50 mg/kg) was performed as described previously (11Hetman M. Filipkowski R.K. Domagala W. Kaczmarek L. Exp. Neurol. 1995; 36: 53-63Crossref Scopus (47) Google Scholar, 12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar, 13Lukasiuk K. Kaczmarek L. Brain Res. 1994; 643: 227-233Crossref PubMed Scopus (36) Google Scholar). Briefly, to exclude effects of the injection itself, the animals were handled and injected with physiological saline daily for 3–4 days before the experimental treatment. The rats were then given either sodium kainate (Sigma) or PTZ (RBI) by intraperitoneal injection and observed for up to 90 min to confirm the occurrence or absence of convulsions. Only the animals displaying clear seizures (that occur a few minutes after PTZ treatment and are initiated at 30–60 min following kainate administration) were used for the experiments (that is, typically in our hands, more than 80% of rats). For collection of the material, rats were decapitated at different times after the drug administration (4–6 animals for each time point), the brains were removed and processed as described in details below. TIMP-LacZ transgenic mice were obtained from Dr. B. R. Williams (14Flenniken A.M. Williams B.R.G. Genes Dev. 1990; 4: 1094-1106Crossref PubMed Scopus (64) Google Scholar). The animals carry the fragment oftimp-1 gene extending from −1373 to +727 (see Fig. 1) fused to LacZ gene encoding reporter enzyme β-galactosidase. Fidelity of the gene construct expression has been documented during development, when patterns of β-galactosidase activity were found to mimic expression of the endogenous timp-1 gene (14Flenniken A.M. Williams B.R.G. Genes Dev. 1990; 4: 1094-1106Crossref PubMed Scopus (64) Google Scholar). Isolated brains were rapidly dissected on ice and the hippocampi were snap-frozen on dry ice. RNA was isolated from frozen tissue according to the procedure of Chomczynski and Sacchi (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) and electrophoresed through 1% agarose gel as described (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar). After blotting onto nylon membranes (Hybond N, Amersham) the filters were prehybridized for 2 h and then hybridized overnight with random primer-labeled probes in Church buffer (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar, 16Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar). For in situ mRNA analysis the procedure described by Konopka et al. (17Konopka D. Nowicka D. Filipkowski R.K. Kaczmarek L. Neurosci. Lett. 1995; 185: 167-170Crossref PubMed Scopus (25) Google Scholar) was followed. Isolated brains were immediately frozen on dry ice. Twenty micrometer cryostat sections were fixed in 4% cold paraformaldehyde in PBS, dehydrated, and prehybridized for 2 h at 37 °C in buffer containing: 50% formamide, 2 × SSC (0.3 m sodium chloride, 0.03m sodium citrate, pH 7,0), 1 × Denhardt's solution (0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 200 μg/ml single-stranded DNA, and 20 mmdithiothreitol. Next, the sections were hybridized overnight in the above solution containing additionally 10% dextran sulfate and35S-labeled cDNA (random primers) probe at 37 °C. The probe was kindly provided by Dr. Y. Citri (5Nedivi E. Hevroni D. Naot D. Israeli D. Citri Y. Nature. 1993; 363: 718-722Crossref PubMed Scopus (437) Google Scholar). The sections were washed for 15 min and then for 60 min in 50% formamide, 2 × SSC at room temperature. Afterward, the sections were exposed against β-Max Hyperfilm (Amersham). The autoradiograms were analyzed with an aid of PC-based computer program Provision. The expression of c-Fos protein was assessed essentially as described before (18Kaminska B. Kaczmarek L. Chaudhuri A. J. Neurosci. 1996; 16: 3968-3978Crossref PubMed Google Scholar). Following the appropriate treatments, as indicated under “Results,” the rats were anaesthetized with chloride hydrate overdose and perfused with saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. The brains were removed and stored in the same fixative for 24 h at 4 °C and then in 30% sucrose with 0.02% sodium azide at 4 °C until needed. The brains were slowly and gradually frozen in a heptane/dry ice bath and sectioned at 20 μm on a cryostat. The sections were washed three times in PBS, pH 7.4, incubated 10 min in 0.3% H2O2 in PBS, washed twice in PBS, then incubated with a polyclonal antibody (anti-c-Fos, 1:1000, Santa Cruz number sc-52) for 48 h at 4 °C in PBS with azide (0.01%) and normal goat serum (3%). After that the sections were washed three times in PBS containing Triton X-100 (0.3%, Sigma), incubated with goat anti-rabbit biotinylated secondary antibody (1:1000, Vector) in PBS/Triton and normal goat serum (3%) for 2 h, washed three times in PBS/Triton, incubated with avidin-biotin complex (1:1000, Vector, in PBS/Triton) for 1 h and washed three times in PBS. The immunostaining reaction was developed using the glucose oxidase-3,3′-diaminobenzidine tetrachloride-nickel method. The sections were incubated in PBS with 3,3′-diaminobenzidine tetrachloride (0.05%), glucose (0.2%), ammonium chloride (0.04%), ammonium nickel sulfate (0.1%) (all from Sigma) for 5 min, then 10% (v/v) glucose oxidase (Sigma, 10 units/ml in H2O) was added. The staining reaction was stopped by two to three washes with PBS. The sections were mounted on gelatin-covered slides, air-dried, dehydrated in ethanol solutions and xylene, and embedded in Entellan (Merck). Brain tissue from the rat hippocampi was extracted and immediately processed on ice for nuclear protein extraction (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar, 18Kaminska B. Kaczmarek L. Chaudhuri A. J. Neurosci. 1996; 16: 3968-3978Crossref PubMed Google Scholar). The tissue was manually pulverized with a Teflon pestle and suspended in 0.5 ml of buffer A (10 mm Hepes, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, and protease inhibitors: 1 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mg/ml pepstatin A) (all products from Sigma). After incubation for 15 min on ice, Nonidet P-40 was added to final concentration of 1% before centrifugation at 12,000 rpm for 1 min at 4 °C. The crude pellet was resuspended in buffer B (20 mm Hepes, 0.84 m NaCl, 1.5 mmMgCl2, 0.4 mm EDTA, 1 mmdithiothreitol, 25% (v/v) glycerol, and protease inhibitors as above) and incubated for 15 min at 4 °C. After centrifugation for 15 min at 12,000 rpm, the supernatant was frozen at −70 °C. The protein content was estimated by the Bradford method and verified by Coomassie staining of SDS-polyacrylamide gel electrophoresis 12% Tris glycine gels. We applied the EMSA technique to assess the DNA binding activities of the extracted nuclear proteins from the different experimental conditions. Fifteen micrograms of nuclear proteins were preincubated for 10 min at room temperature in binding buffer (10 mmHepes, 25 mm KCl, 0.5 mm EDTA, 0.25 μg/ml bovine serum albumin, 1 mm dithiothreitol, 20 μg/ml poly(dI-dC) and subsequently incubated with 40 fmol (30,000–40,000 Cerenkov's cpm) of end-labeled probe for 20 min at room temperature. The timp-1 promoter-derived sequences as well as their mutated variants were used in the experiments (Fig. 1). The probes were labeled with [α-32P]dCTP (Amersham) and purified on Sephadex G-50 spun columns. Following incubation, 2 μl of loading buffer containing 0.3% bromphenol blue, 3% glycerol was added to the samples and electrophoresed at 130 V for 2 h in a nondenaturing 4% polyacrylamide gel. Electrophoresis was performed in a low ionic strength buffer (6.7 mm Tris-HCl, pH 7.5, 1 mmEDTA, and 3.3 mm sodium acetate). Gels were dried and exposed to phosphor screens (Molecular Dynamics) overnight. Gel images were obtained with a PhosphorImager (Molecular Dynamics). To facilitate comparison among the different conditions, the autoradiograms were scanned densitometrically and average gray/pixel level was measured in the area of the band. To identify the components of the AP-1 complex, supershift analysis was applied (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar, 13Lukasiuk K. Kaczmarek L. Brain Res. 1994; 643: 227-233Crossref PubMed Scopus (36) Google Scholar). Commercially available (Santa Cruz) polyclonal antibodies against the following members of AP-1 family were used: c-Fos (sc-52X), FosB (sc-48X), Fra-1 (sc-183X), Fra-2 (sc-171X), JunB (sc-46X), c-Jun (sc-822X), and JunD (sc-74X). All antibodies (1 mg/1 ml) were affinity purified by the manufacturer and had no detectable cross-reactivity with other members of the Fos and Jun families. This was confirmed by Western blot analysis (see Refs. 12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar and 18Kaminska B. Kaczmarek L. Chaudhuri A. J. Neurosci. 1996; 16: 3968-3978Crossref PubMed Google Scholar). One microliter of each antibody was added to 10 μl of reaction volume containing the nuclear protein extract (10 μg) and incubated for 1 h at 4 °C. Afterward, the labeled oligonucleotide was added to the reaction mixture and the EMSA protocol was followed, as described above. The samples were then electrophoresed at 110 V for 5 h with recirculation of the electrophoresis buffer. Gels were dried and exposed to phosphor screens and analyzed with a PhosphorImager as described above. Primary cultures of dentate gyrus cells were obtained from 5-day-old rat pups using a modification of procedure described previously (19Figiel I. Kaczmarek L. Neurochem. Int. 1997; 31: 229-240Crossref PubMed Scopus (34) Google Scholar). Briefly, hippocampal tissue was sliced mechanically and placed in Krebs-Ringer bicarbonate medium supplemented with 3 mg/ml bovine serum albumin and 1.2 mm MgSO4 (solution A). The area dentata was then dissected from each slice and transferred to a tube containing the same solution. After a short centrifugation at 150 × g, the tissue was resuspended in 5 ml of the solution A containing 0.05 mg/ml trypsin (Life Technologies, Inc.), and left in a rotary bath at 37 °C (200 rpm) for 20 min. Next, 5 ml of solution A, containing 12.8 μg/ml DNase I (Sigma) and 83 μg/ml soybean trypsin inhibitor was added to the suspension, which was immediately centrifuged. The pellet was resuspended in 3 ml of the same solution, containing in addition 80 μg/ml DNase I, 0.52 μg/ml trypsin inhibitor, and 2.7 mm MgSO4. The tissue was dissociated with a Pasteur pipette, sedimented for 15 min, and the pellet was redissociated as above in 2 ml. The two supernatants were collected and supplemented with 3 ml of Krebs-Ringer biocarbonate medium containing 3 mg/ml bovine serum albumin, 2.4 mmMgSO4, and 0.1 μm CaCl2. After centrifugation for 10 min at 150 × g the pellet was resuspended in the culture medium. Cells were plated on poly-l-lysine-coated glass coverslips at a density of 150,000 per coverslip. Cultures were maintained for 24 h in Dulbecco's modified Eagle's (DMEM, Life Technologies, Inc.) medium containing 10% fetal calf serum (with 25 mm KCl, 2 mm glutamine and penicillin (50 units/ml)/streptomycin (50 μg/ml)). Twenty-four h later the cultures were transferred into the chemically defined medium consisting of DMEM supplemented with 1 × N-2 Supplement (Life Technologies, Inc.), 25 mm KCl, 2 mm glutamine, and penicillin (50 units/ml)/streptomycin (50 μg/ml). Cells grown for 4 days in vitro were transfected using the calcium-phosphate procedure. A day later, the cells were exposed to glutamate (0.1 mm). The gene constructs used in this study to assess thetimp-1 AP-1 activity in cultured dentate gyrus neurons are schematically depicted in the Fig. 1. The genes were constructed from fragments of wild type timp-1promoter or promoter mutated in AP-1 site that were obtained by polymerase chain reaction with prT-61CAT and prTmut(AP1)CAT plasmids (8Bugno M. Greave L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordula T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar) used as templates for reactions. For all reactions the same pair of polymerase chain reaction primers were used: 5′-gcagttctactcgagcttgcatgcctgcaggtcga-3′ and 5′-ggctaggatcctgaaaatctcgccaagtctgtcgag-3′. Both, p61PTimp1(AP1)GFP and p61Ptimp1(AP1mut)GFP, were generated by subcloning the aforementioned polymerase chain reaction products digested with XhoI-BamHI into theXhoI-BamHI sites of pEGFP-1 plasmid (CLONTECH). All constructs were verified by sequencing. prT-61CAT and prTmut(AP1)CAT plasmids were kindly provided by Dr. T. Kordula (8Bugno M. Greave L. Gatsios P. Koj A. Heinrich P.C. Travis J. Kordula T. Nucleic Acids Res. 1995; 23: 5041-5047Crossref PubMed Scopus (101) Google Scholar). For each transfection 3 μg of total plasmid DNA per coverslip were used. Usually a 2:1 ratio of timp-1 promoter containing vector p61PTimp1(AP1)GFP or p61Ptimp1(AP1mut)GFP to pSVβgal (used for assessing the transfection efficiency during the normalization steps of evaluation of the results) was used. All plasmids were purified using EndoFree Plasmid Maxi Kit (QIAgen). Neurons from dentate gyrus of hippocampus were transfected by the calcium phosphate method described earlier (20Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar) with modifications. Cells growing for 4–5 days were used for transfection. The culture medium was removed and saved. The cells growing on coverslips were moved into a 24-well dish (one coverslip per each well) and incubated for 1 h in 300 μl of preheated DMEM containing 2 mmsodium kynurenate, 10 mm MgCl2. During this time DNA/calcium phosphate precipitate was prepared. One volume of DNA in 0.5 m CaCl2 was added dropwise to the 1 volume of 2 × Hepes-buffered saline (2 × HBS: 274 mm NaCl, 10 mm KCl, 1.4 mmNa2HPO4·7H2O, 15 mmd-glucose, 42 mm HEPES, pH 7.08) and the mixture was left for 30 min in room temperature to let the precipitate form. After 30 min of precipitate formation, 33 μl of mixture was added to the cells. Cultures were incubated with DNA/calcium phosphate precipitate for 35 min. After incubation, the cells were washed once with DMEM, 2 mm sodium kynurenate, 10 mm MgCl2, and twice with DMEM. The saved conditioned medium was mixed 1:1 with new medium and added back to the cells. Twenty-four h after the transfection, the cells were exposed to 0.1 mm glutamate. After 24-h cultures were analyzed under the fluorescent microscope to score for GFP-positive cells. Afterward, cultures were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside to score for β-galactosidase transfectants. For normalization purposes (see above) the pSVβgal co-transfected cells were washed once with PBS and fixed for 5 min in 1% formaldehyde, 0.2% glutaraldehyde in PBS. Then the cells were incubated overnight at 37 οC in 0.8 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, 4 mm K3Fe(CN)6, 4 mmK4Fe(CN)6, 4 mmMgCl2·6H2O in PBS. After incubation, the blue-stained cells were counted. Brains were cut into 20 μm-thick sections and loaded on gelatin-coated slides and fixed in for 5 min in 1% formaldehyde, 0.2% glutaraldehyde in PBS at 4 οC. Then the sections were rinsed in PBS twice and incubated overnight in 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, 5 mm K3Fe(CN)6, 5 mmK4Fe(CN)6, 2 mmMgCl2·6H2O in PBS at 37 οC. Sections were dehydrated and embedded in Entellan. In the first experiment we analyzed, with Northern blot technique, the time course of timp-1 mRNA accumulation in the hippocampus after treating rats with kainate. We found significant increases intimp-1 mRNA at 6–24 h after the treatment (Fig.2 A). Notably, using exactly the same conditions as previously reported, the mRNA level of c-fos as well as other AP-1 components peak at 1–6 h (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar). The kainate-evoked increase in timp-1 mRNA accumulation at 6 h was greatly suppressed by pretreatment of the rats with the protein synthesis inhibitor, cycloheximide (Fig.2 B), suggesting that a significant component oftimp-1 mRNA accumulation requires precedingde novo protein synthesis. To extend the observation of seizure-driven timp-1 mRNA accumulation, and to exclude the possibility that our observations are solely linked to kainate neurotoxicity, we employed another seizure model that involves an application of PTZ, known to produce massive neuronal excitation in the hippocampus without, however, any adverse effects on neuronal survival. Again, a significant increase intimp-1 mRNA levels was observed after the PTZ-evoked seizures, with peak values of timp-1 mRNA levels at 2 h following treatment. Importantly, this elevation was slightly delayed in comparison with c-fos mRNA, that peaked at 45 min after the PTZ injection (Fig. 2C). We also analyzed the spatial pattern of timp-1 mRNA expression at either 6 h after kainate administration or 2 h following PTZ treatment. The in situ hybridization autoradiograms were compared with c-Fos immunocytochemistry performed on parallel sections. Fig. 3 presents the striking resemblance between timp-1 mRNA and c-Fos protein distribution patterns. In the hippocampus of PTZ-treated animal, both timp-1 mRNA and c-Fos protein are most abundant in the dentate gyrus, whereas in the kainate-treated rat expression of both timp-1 and c-Fos occurs throughout the neuronal cell body layer of all hippocampal subfields. To investigate whether timp-1 promoter can be activated following seizures and thus to exclude that timp-1 mRNA accumulation might be solely derived from the increased messenger stability, we employed transgenic mice harboring a gene construct containing the timp-1 regulatory region fused to β-galactosidase coding region (see Fig. 1). The mice were treated with kainate (20 mg/kg) and 24 h later their brains were analyzed for β-galactosidase activity. Fig. 4shows that kainate administration resulted in a marked stimulation of the timp-1 promoter, most notably in the dentate gyrus. A similar, albeit less pronounced, effect was also observed in animals treated with PTZ (50 mg/kg, not shown). In the next series of experiments, we used synthetic oligonucleotides carrying timp-1 gene regulatory sequences spanning the promoter region from −66 to −35 (see Fig. 1), to investigate transcription factors binding before and after either kainate or PTZ treatment. In the first experiment, we investigated the pattern of DNA binding at different times after treatment with kainate. Three major bands of DNA binding could be recognized. Fig.5 A shows the strong binding that was observed in the band designated as I and that was markedly induced at 6 h after kainate administration, i.e. when the timp-1 mRNA starts to peak and thus when one could expect the gene transcription to be active. The intensity of the binding decreased at 24 h following treatment (not shown). In our previous study using a consensus AP-1 sequence as a probe for DNA binding experiments, we reported that 2–6 h after kainate exposure the AP-1 complex is composed predominantly of c-Fos, FosB, JunB, and JunD proteins (12Kaminska B. Filipkowski R.K. Zurkowska G. Lason W. Przewlocki R. Kaczmarek L. Eur. J. Neurosci. 1994; 6: 1558-1566Crossref PubMed Scopus (113) Google Scholar). Using the AP-1/Stat/Ets sequence derived from thetimp-1 promoter, we found similar AP-1 DNA-binding proteins, including phosphorylated c-Jun (Fig. 5 B). Next, we used wild type (wt) as well as variants of the timp-1 regulatory region, mutated specifically at either AP-1 (mutAP-1) or Stat/Ets (mutStat/Ets) element, to identify its binding proteins present in rat hippocampus 6 h after KA administration. Fig. 5 C shows that a mutation in the AP-1 element completely prevented the kainate-evoked band I protein binding, whereas mutations in Stat/Ets region did not affect any of the bands. Furthermore, we carried out competition experiments in which the hippocampal extracts, obtained from animals at 6 h after kainate, were preincubated with an excess of unlabeled timp-1 regulatory region (either wt or mut) before adding the wild type 32P-labeled probe. Fig.5 C documents that competition with the wild type probe abolishes DNA binding, most notably in bands I and II. Similar effects were also observed in the case of a probe mutated within the Stat/Ets element. In contrast, oligonucleotides mutated in the AP-1 site only weakly affected binding to the wild type probe (Fig.5 C). Essentially the same pattern, indicating requirement for intact AP-1 site of DNA binding of the timp-1 promoter region, was observed at 2 h after treating rats with pentylenetetrazole,i.e. at the time coinciding with timp-1mRNA accumulation in this seizure model (Fig. 5 D). In the previous experiments we found the most robust timp-1mRNA accumulation in hippocampal dentate gyrus granule cell neuronal layer, in response to markedly elevated neuronal excitation. To further investigate the role of timp-1 AP-1 regulatory sequence in excitatory amino acid-driven gene expression we turned to glutamate-stimulated dentate gyrus cells in culture (19Figiel I. Kaczmarek L. Neurochem. Int. 1997; 31: 229-240Crossref PubMed Scopus (34) Google Scholar). These cultures derived from 5-day-old rat pups are composed of neuronal and glial elements. The cultures were transfected with a modified calcium phosphate technique (see “Exp
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