Abstract
23 min readThe transcription factor, AplysiaCCAAT enhancer-binding protein (ApC/EBP), plays a crucial role in long term facilitation, a synaptic mechanism of long term memory in Aplysia. To gain a clue to whether the mammalian C/EBP family of transcription factors are also involved in long term memory, we examined how C/EBP activities in hippocampal neurons can be modulated in response to cAMP and Ca2+, crucial inductive signals for memory formation. As a result, stimulation of either cAMP or Ca2+ signals in hippocampal neurons was found to enhance mRNA expressions and DNA binding activities of C/EBPβ and C/EBPδ. Furthermore, it is indicated that CaM kinases have essential roles for increasing the expression and DNA binding activities of C/EBPβ in hippocampal neurons activated by membrane depolarization. Overexpression of constitutively active calcium/calmodulin-dependent kinase IV was found to directly stimulate either C/EBPβ-dependent or C/EBPδ-dependent transcription, reinforcing the evidence that C/EBP family members contribute to Ca2+-dependent transcription. Thus, these results suggest that C/EBPβ and C/EBPδ may be involved in the transcription-dependent phase of memory formation by increasing the expression of both the DNA binding and the transcriptional activities under the direction of cAMP and/or Ca2+signaling in hippocampal neurons. The transcription factor, AplysiaCCAAT enhancer-binding protein (ApC/EBP), plays a crucial role in long term facilitation, a synaptic mechanism of long term memory in Aplysia. To gain a clue to whether the mammalian C/EBP family of transcription factors are also involved in long term memory, we examined how C/EBP activities in hippocampal neurons can be modulated in response to cAMP and Ca2+, crucial inductive signals for memory formation. As a result, stimulation of either cAMP or Ca2+ signals in hippocampal neurons was found to enhance mRNA expressions and DNA binding activities of C/EBPβ and C/EBPδ. Furthermore, it is indicated that CaM kinases have essential roles for increasing the expression and DNA binding activities of C/EBPβ in hippocampal neurons activated by membrane depolarization. Overexpression of constitutively active calcium/calmodulin-dependent kinase IV was found to directly stimulate either C/EBPβ-dependent or C/EBPδ-dependent transcription, reinforcing the evidence that C/EBP family members contribute to Ca2+-dependent transcription. Thus, these results suggest that C/EBPβ and C/EBPδ may be involved in the transcription-dependent phase of memory formation by increasing the expression of both the DNA binding and the transcriptional activities under the direction of cAMP and/or Ca2+signaling in hippocampal neurons. Ca2+/cAMP-responsive element-binding protein Ca2+/cAMP-responsive element CCAAT/enhancer-binding protein Ca2+/calmodulin-dependent protein kinase Ca2+/calmodulin-dependent protein kinase IV Ca2+/calmodulin-dependent protein kinase II activating transcription factor electrophoretic mobility shift assay phosphate-buffered saline base pair(s) Aplysia C/EBP adenosine 3′,5′-cyclic phosphorothiolate-Sp. Memory has two phases: short term memory and long term memory. A number of pharmacological studies have demonstrated that the stabilization of long term memory requires the synthesis of new proteins and RNAs (1Davis H.P. Squire L.R. Psychol. Bull. 1984; 96: 518-559Crossref PubMed Scopus (1268) Google Scholar, 2Matthies H. Prog. Neurobiol. 1989; 32: 277-349Crossref PubMed Scopus (189) Google Scholar). This requirement suggests that transcription is critical for this process. Among a number of transcription factors expressed in neuronal cell nuclei, the Ca2+/cAMP-responsive element-binding protein (CREB)1 has been implicated as being essential to long term memory of Aplysia, Drosophila, and mice (3Dash P.K. Hochner B. Kandel E.R. Nature. 1990; 345: 718-721Crossref PubMed Scopus (593) Google Scholar, 4Bartsch D. Ghirardi M. Skehel P.A. Karl K.A. Herder S.P. Chen M. Bailey C.H. Kandel E.R. Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (497) Google Scholar, 5Yin J.C. Wallach J.S. Del Vecchio M. Wilder E.L. Zhou H. Quinn W.G. Tully T. Cell. 1994; 79: 49-58Abstract Full Text PDF PubMed Scopus (836) Google Scholar, 6Yin J.C. Del Vecchio M. Zhou H. Tully T. Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (568) Google Scholar, 7Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1587) Google Scholar). InAplysia, long term facilitation is a basic synaptic mechanism for a non-associative learning response. The long term synaptic modification is characterized by a consolidation period during which gene expression is required. During this phase, the transcription factor Aplysia CCAAT enhancer-binding protein (ApC/EBP) is found to be increasingly induced in response to cAMP signals (8Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (497) Google Scholar). There is a CRE site in the 5′-untranslated region of ApC/EBP, suggesting that CREB can control the expression of ApC/EBP (8Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (497) Google Scholar). Furthermore, blocking the function of ApC/EBP either by antisense oligonucleotides or by specific antibody inhibits long term facilitation selectively without affecting the short term processes (8Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (497) Google Scholar). These data indicate that induction of another transcription factor ApC/EBP by CREB is also essential for long term facilitation, in addition to CREB activation (8Alberini C.M. Ghirardi M. Metz R. Kandel E.R. Cell. 1994; 76: 1099-1114Abstract Full Text PDF PubMed Scopus (497) Google Scholar). In mammals, seven members of C/EBP family of transcription factors have been cloned molecularly and their biological roles extensively analyzed in a variety of systems (9Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar, 10Akira S. Kishimoto T. Immunol. Rev. 1992; 127: 25-50Crossref PubMed Scopus (471) Google Scholar, 11Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar, 12Metz R. Ziff E. Genes Dev. 1991; 5: 1754-1766Crossref PubMed Scopus (302) Google Scholar, 13Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. Genes Dev. 1990; 4: 1541-1551Crossref PubMed Scopus (420) Google Scholar, 14Friedman A.D. Landschulz W.N. McKnight S.L. Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (363) Google Scholar, 15Landschulz W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes Dev. 1988; 2: 786-800Crossref PubMed Scopus (630) Google Scholar, 16MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (943) Google Scholar, 17Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (459) Google Scholar, 18Roman C. Platero J.S. Shuman J.D. Calame K. Genes Dev. 1990; 4: 1404-1415Crossref PubMed Scopus (191) Google Scholar, 19Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar, 20Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (439) Google Scholar, 21Xu L.X. Wan D.F. Liu Y.F. Li H.N. Zhang P.P. Sui Y.F. Gu J.R. Chung Hua I Hsueh Tsa Chih. 1996; 76: 20-23Google Scholar, 22Yamanaka R. Kim G.-D. Radomska H.S. Lekstorm-Himes J. Smith L.T. Antonson P. Tenen D.G. Xanthopoulos K.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6462-6467Crossref PubMed Scopus (153) Google Scholar, 23Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (838) Google Scholar, 24Tanaka T. Akira S. Yoshida K. Umemoto M. Yoneda Y. Shirafuji H. Fujisawa H. Suematu S. Yoshida K. Kishimoto T. Cell. 1995; 80: 353-361Abstract Full Text PDF PubMed Scopus (472) Google Scholar, 25Screpanti T. Romani L. Musiani P. Modesti A. Fattori E. Lazzaro D. Sellito C. Scarpa S. Bellavia D. Lattanzio G. Bistoni F. Frati L. Cortese R. Gulino A. Ciliberto G. Costantini F. Poli V. EMBO J. 1995; 14: 1932-1941Crossref PubMed Scopus (376) Google Scholar, 26Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (645) Google Scholar). However, much less is known about the expression and function of C/EBP family members in the neurons of mammalian brain, except that C/EBPα mRNA was detected in mouse hippocampus, cerebellum, and cortex by in situ hybridization (27Kuo C.F. Xanthopoulos K.G. Darnell Jr., J.E. Development. 1990; 109: 473-481Crossref PubMed Google Scholar). It was recently shown that two CRE sites in the core promoter of C/EBPβ gene are mostly recognized by CREB in liver and in several cell lines (28Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar). Transfection experiments with promoter constructs where the CREB sites were mutated further indicated that these sites are important to maintain both basal promoter activity and C/EBPβ inducibility through CREB (28Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar). However, in several brain regions including the hippocampus, little information is available about the induction of C/EBP family members by cAMP and Ca2+, both of which are known to activate CREB in neuronal cells such as hippocampal neurons (29Frey U. Huang Y.-Y. Kandel E.R. Science. 1993; 260: 1661-1664Crossref PubMed Scopus (1016) Google Scholar, 30Moore A.N. Waxham M.N. Dash P.K. J. Biol. Chem. 1996; 271: 14214-14220Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 31Impey S. Mark M. Villacres E.C. Poser S. Chavkin C. Storm D.R. Neuron. 1996; 16: 973-982Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 32Deisserroth K. Bito H. Tsien R.W. Neuron. 1996; 16: 89-101Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 33Bito H. Deisserroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar). The hippocampus in mammalian brain is involved in the normal formation of long term declarative memory (34Squire L.R. Psych. Rev. 1992; 99: 195-231Crossref PubMed Scopus (4124) Google Scholar). Here, we have found that a prime C/EBP transcription factor in the hippocampus is C/EBPβ. To clarify whether C/EBPβ activity in hippocampal neurons is further modulated by the stimulation of cAMP or Ca2+ signals, which are crucial inducers for memory formation (3Dash P.K. Hochner B. Kandel E.R. Nature. 1990; 345: 718-721Crossref PubMed Scopus (593) Google Scholar, 4Bartsch D. Ghirardi M. Skehel P.A. Karl K.A. Herder S.P. Chen M. Bailey C.H. Kandel E.R. Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (497) Google Scholar, 5Yin J.C. Wallach J.S. Del Vecchio M. Wilder E.L. Zhou H. Quinn W.G. Tully T. Cell. 1994; 79: 49-58Abstract Full Text PDF PubMed Scopus (836) Google Scholar, 6Yin J.C. Del Vecchio M. Zhou H. Tully T. Cell. 1995; 81: 107-115Abstract Full Text PDF PubMed Scopus (568) Google Scholar, 7Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1587) Google Scholar, 35Tully T. Bolwig G. Christensen J. Connolly M. Vecchio M.D. DeZazzo J. Dubnau J. Jones C. Pinto S. Regulski M. Svedberg B. Velinzon K. Function & Dysfunction in the Nervous System. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 207-218Google Scholar, 36Silva A.J. Paylor R. Wehner J.M. Tonegawa S. Science. 1992; 257: 206-211Crossref PubMed Scopus (1085) Google Scholar, 37Bach M.E. Hawkins R.D. Osman M. Kandel E.R. Mayford M. Cell. 1995; 81: 905-915Abstract Full Text PDF PubMed Scopus (386) Google Scholar, 38Tsien J.Z. Huerta P. Tonegawa S. Cell. 1996; 87: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (1453) Google Scholar, 39Abel T. Nguyen V.P. Barad M. Deuel A.S.T. Kandel E.R. Bourtchuladze R. Cell. 1997; 88: 615-626Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar), we used cultured hippocampal neurons for detailed biochemical analyses (40Baranes D. Lopez-Garcia J.C. Chen M. Bailey C.H. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4706-4711Crossref PubMed Scopus (46) Google Scholar). We have found that the expression and DNA binding activities of C/EBPβ and δ are enhanced by the stimulation of cAMP or Ca2+ signals in cultured hippocampal neurons. Our results also suggest that CaMKIV activated by Ca2+ signal not only induces expression of C/EBP members, but also directly enhances C/EBP-dependent gene transcriptions. Therefore, our study supports the possibility that both C/EBPβ and C/EBPδ may be involved in long term plasticity in mammalian brain. Hippocampal neuron cultures were done as described by Baranes et al. (40Baranes D. Lopez-Garcia J.C. Chen M. Bailey C.H. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4706-4711Crossref PubMed Scopus (46) Google Scholar). To stimulate cAMP signals, water-soluble forskolin (50 μm; Research Biochemicals International) was added to 14-day-old cultures. To induce membrane depolarization of hippocampal neurons in the culture, the cultures were treated as described by Bito et al. (33Bito H. Deisserroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar). When used, kinase inhibitor KN93 (30 μm, Calbiochem), KN92 (30 μm, Calbiochem), and KN62 (10 μm, Calbiochem) were present during the preincubation period of 30 min prior to depolarization and during depolarization. Effects of cAMP signal stimulation and membrane depolarization on gene expressions or DNA binding activities were measured 3–4 h after stimulation. Rat hippocampal neurons were grown on poly-d-lysine- and laminin-coated 12-mm glass coverslips in 35-mm dishes (Corning) under the same conditions described by Baranes et al. (40Baranes D. Lopez-Garcia J.C. Chen M. Bailey C.H. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4706-4711Crossref PubMed Scopus (46) Google Scholar). Cells were washed with Tyrode's solution (37 °C) once and fixed with 4% paraformaldehyde solution for 15 min at 37 °C. They were then washed with PBS three times and permeabilized with 0.25% Triton X-100 in PBS at 37 °C for 5 min, and the nonspecific bindings were blocked with 10% goat serum, 0.1% Triton X-100, 20 mm glycine in PBS at 37 °C for 30 min. Double immunolabeling was performed by incubating cells overnight with a mouse monoclonal anti-MAP2 antibody (Sigma, 1:100) and rabbit polyclonal anti-C/EBPβ or anti-C/EBPδ antibodies (Santa Cruz, 1 μg/ml). Following three washes with PBS, the cells were incubated with Cy3-conjugated goat anti-mouse IgG (Cedarlane Laboratories Ltd, 1:100) and fluorescein-conjugated goat anti-rabbit IgG (Cedarlane Laboratories Ltd, 1:100) in PBS at room temperature for 1 h. The coverslips were washed five times with PBS, mounted, and examined by fluoromicroscopy. (Sp)-cAMPS stimulation of hippocampal slices was done as described by Huang et al. (41Huang Y.Y. Li X.-C. Kandel E.R. Cell. 1994; 79: 69-79Abstract Full Text PDF PubMed Scopus (440) Google Scholar). Hippocampal slices were incubated in perfusion solutions containing 50 μm(Sp)-cAMPS ((Sp)-cAMPS stimulation) or perfusion solutions without (Sp)-cAMPS (control) for 30 min. After 30 min of incubation, slices were maintained in the perfusion solution for 2 h. Then, RNAs were prepared from those slices for RNase protection assay. A 220-bp rat C/EBPβ cDNANcoI fragment, 342-bp mouse C/EBPβ cDNANcoI-PstI fragment covering the leucine-zipper domain, 298-bp mouse C/EBPδ cDNA NcoI-XhoI fragment, and 490-bp rat C/EBPα cDNANcoI-NotI fragment were subcloned into pBluescript SK (Stratagene). Templates for preparing Zif268 cRNA (180 bp is protected), actin cRNA (250 bp), and cyclophilin cRNA probe (100 bp) were purchased from Ambion. Antisense cRNA probes were synthesized as described previously (42Yukawa K. Yasui T. Yamamoto A. Shiku H. Kishimoto T. Kikutani H. Gene (Amst.). 1993; 133: 163-169Crossref PubMed Scopus (33) Google Scholar). Total RNAs were prepared from tissues or cultured hippocampal neurons using the acid phenol extraction method of Chomczynski and Sacchi (43Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) with RNAzol™ B solution purchased from Biotecx Laboratories, Inc. and performed according to manufacturer's protocol. RNase protection assays were performed by hybridizing 25 μg of total RNA from the hippocampus or 2.5 μg of total RNA from cultured hippocampal neurons with 1 × 105 cpm of each labeled cRNA at 68 °C for more than 1 h. RNase digestion and analysis were performed as described previously (42Yukawa K. Yasui T. Yamamoto A. Shiku H. Kishimoto T. Kikutani H. Gene (Amst.). 1993; 133: 163-169Crossref PubMed Scopus (33) Google Scholar). For Western blot analysis, nuclear extracts prepared from cultured hippocampal neurons for gel shift assay were used. Twenty micrograms of each sample were adjusted to give a final solution of 60 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 5% β-mercaptoethanol, heated at 100 °C for 5 min, electrophoresed through 10% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membrane (Millipore). C/EBPβ was detected with the ECL Western blotting detection system as instructed by the manufacturer (Amersham). The dilution factor for anti-C/EBPβ antibody was 1:100 (Santa Cruz). Nuclear extracts of mice hippocampus were prepared as described by others (30Moore A.N. Waxham M.N. Dash P.K. J. Biol. Chem. 1996; 271: 14214-14220Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) with some modifications. The tissues were homogenized (10 strokes) in four volumes of a buffer containing 0.25 m sucrose, 15 mm Tris-HCl, pH 7.6, 60 mm KCl, 15 mm NaCl, 5 mmEDTA, 1 mm EGTA, and protease inhibitors (1 × α-Complete™ from Boehringer Mannheim, 0.5 mm phenylmethylsulfonyl fluoride, and 1 mmdithiothreitol) in a Dounce homogenizer. After centrifugation, the pelletized material was resuspended in buffer A (10 mmHEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1× α-Complete, 0.5 mmphenylmethylsulfonyl fluoride, and 1 mm dithiothreitol) by gently pipetting and kept on ice for 15 min. Nonidet P-40 was added to reach a final concentration of 0.6% and vortexed for 10 s. After centrifugation for 60 s, the nuclei were resuspended in 70 μl of buffer C (20 mm HEPES, pH 7.9, 25% glycerol, 0.4m NaCl, 1 mm EDTA, 1 mm EGTA, 1× α-Complete, 0.5 mm phenylmethylsulfonyl fluoride, and 1mm dithiothreitol) and rocked on an Eppendorf shaker for 20 min at 4 °C. The supernatant solution after centrifugation was frozen as nuclear extract. To prepare nuclear extracts from hippocampal neurons in culture, cells were washed with Tyrode's solution once, then immersed with 300 μl of solution A, scraped, and collected into Eppendorf tubes. The following procedure is identical to the above for preparation of tissue nuclear extracts. Protein amounts were quantified by the colorimetric determination using bicinchoninic acid solution (Sigma). For electrophoretic mobility shift assay (EMSA), C/EBP consensus oligonucleotide, 5′-TGCAGATTGCGCAATCTGCA-3′ (Santa Cruz), and the core promoter sequence of C/EBPβ gene, 5′-GCGGCCGGGCAATGACGCGCACCG-3′ (28Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar), were 32P-labeled with [γ-32P]ATP, using T4 polynucleotide kinase. Labeled DNA was incubated with 5 μg of nuclear extract protein at room temperature for 20 min in the binding buffer containing 10 mm Tris, pH 7.5, 50 mm NaCl, 1 mmEDTA, 5% glycerol, and 1 μg of poly(dI-dC). DNA-protein complexes were resolved as described previously (44Yukawa K. Butz K. Yasui T. Kikutani H. Hoppe-Seyler F. J. Virol. 1996; 70: 10-16Crossref PubMed Google Scholar). Supershift assays were performed as described above with the exception that, after the incubation of probes with extracts, 1 μg of antibody (TransCruz™ gel supershift antibodies to C/EBPα, β, δ, CRP1, Rb, CREB1, ATF3, and ATF4; Santa Cruz) was added and incubated for another 30 min at room temperature. Expression vectors for C/EBPβ and δ (gift from Dr. P. F. Johnson) and luciferase reporter vector with four tandem C/EBP binding sites were transfected into HeLa cells with or without the construct for expressing the constitutively active form of CaMKIV (45Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (649) Google Scholar) through CaPO4 method (Transfection MBS mammalian transfection kit, Stratagene). Triplicate transfections were done in three independent experiments. Two days after transfection, cells were harvested for luciferase assay and their luciferase activities were examined using the Dual-luciferase™reporter assay system (Promega). In the system, the luciferase activities of the reporter were normalized to the Renillaluciferase activities from 20 ng of the cotransfected pRL-SV40 (Promega). The experimental results were represented as the mean ± S.E. of the values, which were calculated by comparing the activities of cotransfection experiments with those obtained by the transfections of the luciferase reporter vector alone. To know whether any C/EBP family member is expressed in the hippocampus, which is a crucial region for explicit memory formation in vertebrates, we examined the mRNA expressions of C/EBP members in hippocampus by performing RNase protection assay. The result indicated that C/EBPβ and δ transcripts are expressed in the mouse hippocampus (Fig. 1 A). It is also shown that C/EBPβ mRNA is more highly expressed in the mouse hippocampus than C/EBPδ mRNA (Fig. 1 A). Next, we asked which members of C/EBP family is most active in mouse hippocampus by performing EMSA using high affinity C/EBP binding site as a probe. A retarded band was observed in mouse hippocampus nuclear extracts (Fig. 1 B). With the addition of anti-C/EBPβ antibodies to the binding reaction, the result was a prominent supershift of the retarded band, which was removed by the addition of cold C/EBP binding sites as a competitor (Fig. 1 B). Antibodies to C/EBPδ and α also induced supershifts in EMSA of hippocampus, which are much weaker than the supershift induced by anti-C/EBPβ antibodies (Fig. 1 B). Addition of the antibodies to CRP1, another newly identified C/EBP member to the binding reaction could not induce any supershift in EMSA suggesting that CRP1 is not a component of hippocampal C/EBP (Fig. 1 B). Thus, we find that C/EBPβ is a prime C/EBP in the mouse hippocampus. To examine whether C/EBPβ is exactly expressed in hippocampal neurons, double immunofluorescence analyses were performed on cultured hippocampal neurons using antibodies to C/EBP family members and anti-MAP2 antibody to locate neurons. As a result, C/EBPβ was found to be specifically expressed in hippocampal neurons since positive staining was only detected in the nuclei of MAP2 positive cells (Fig. 2, A and B). C/EBPδ is also found to be expressed in some hippocampal neurons (Fig. 2, C and D). However, other neurons scarcely express C/EBPδ. C/EBPδ is found to be rather highly expressed in astrocytes, which are easily distinguished from neurons by remaining unstained by MAP2 (Fig. 2, C and D). This fact is consistent with the previous findings of others (46Yano S. Fukunaga K. Takiguchi M. Ushio Y. Mori M. Miyamoto E. J. Biol. Chem. 1996; 271: 23520-23527Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A small region containing two CREB sites in the C/EBPβ promoter is found to be important in controlling transcription of C/EBPβ gene in liver and several cell lines (28Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar). To examine the possibility that CREB can also control the expression of C/EBPβ even in hippocampus and cultured hippocampal neurons, EMSA of nuclear extracts from hippocampus and cultured hippocampal neurons were performed using the region containing first CREB site of C/EBPβ promoter as a probe. Several retarded bands could be observed in this EMSA suggesting that nuclear factors are actually binding to the region of the C/EBPβ promoter. Furthermore, the addition of CREB antibody to the EMSA reaction could induce a supershift of the major retarded band demonstrating that main binding activity to the C/EBPβ promoter region is due to CREB (Fig. 3). Further addition of cold C/EBPβ promoter probes as competitors weakened the intensity of supershifted bands, thereby demonstrating specific binding reactions in EMSA. The addition of antibodies to other ATF/CREB family members, such as activating transcription factor 3 (ATF3) and activating transcription factor 4 (ATF4), into the EMSA binding reaction could not supershift retarded bands. These results suggest that CREB binds to the crucial region of C/EBPβ promoter and controls the expression of C/EBPβ gene in hippocampal neurons. To know whether CREB activated by cAMP signaling can actually control the expression of C/EBPβ in hippocampal neurons, cultured hippocampal neurons were stimulated by the direct adenylate cyclase activator forskolin and changes of C/EBPβ transcripts were followed by RNase protection assay. After 4 h of exposure to forskolin, C/EBPβ mRNA was found to be prominently induced in cultured hippocampal neurons (Fig. 4 A). The increase of C/EBPβ transcript was observed even from 30 min after addition of forskolin to the culture (data not shown). Consistent with the induction of mRNA, immunoblot analysis with anti-C/EBPβ antibodies showed that the amount of both the 39- and 33-kDa form of C/EBPβ are increased 4 h after forskolin treatment of the culture (Fig. 4 B). To examine whether the increase of C/EBPβ mRNA and protein can enhance its DNA binding activity to C/EBP sites, EMSA was performed using both nuclear extract from culture treated with forskolin for 4 h and nuclear extract from control culture. Forskolin treatment of the culture was found to increase the binding of C/EBP family members to the C/EBP site in EMSA (Fig. 4 C, lanes 1 and 2). Anti-C/EBPβ antibodies induced a supershift, and the amount of these supershifted bands were greatly enhanced by forskolin treatment of the culture (Fig. 4 C). A supershift by anti-C/EBPδ antibodies was also found to be increased by forskolin treatment of the culture (Fig. 4 C). Unrelated antibodies such as anti-Rb antibodies do not induce any supershifts, and the addition of cold competitors almost erased the shifted bands demonstrating specificity of this EMSA. Thus, it is clear that binding of C/EBPβ to C/EBP binding sites is intensified in cultured hippocampal neurons after forskolin treatment. (Sp)-cAMPS is known to stimulate cAMP signaling in hippocampal slices (29Frey U. Huang Y.-Y. Kandel E.R. Science. 1993; 260: 1661-1664Crossref PubMed Scopus (1016) Google Scholar, 41Huang Y.Y. Li X.-C. Kandel E.R. Cell. 1994; 79: 69-79Abstract Full Text PDF PubMed Scopus (440) Google Scholar), which preserve the anatomical relation of neurons in the intact hippocampus. To examine whether stimulation of cAMP signaling can induce the mRNA expression of C/EBP family members even in hippocampal slices, rat and mouse hippocampal slices were treated with (Sp)-cAMPS for 30 min. Two hours after the 30 min of (Sp)-cAMPS treatment, RNAs were prepared from slices and used for RNase protection assay. Both C/EBPβ and δ mRNAs were induced by (Sp)-cAMPS in mouse hippocampal slices (Fig. 5 A). In rat slices, both C/EBPβ and α mRNAs are induced after (Sp)-cAMPS treatment (Fig. 5 B). Thus, the increase of C/EBPβ and δ mRNA can be induced by the stimulation of cAMP signaling pathway, even in hippocampal slices. This result suggests that cAMP signal can induce mRNA expressions of C/EBP family members in intact neurons of hippocampal slices. Previous studies indicated that Ca2+/calmodulin (CaM)-regulated systems can control CREB activity in hippocampal neurons (32Deisserroth K. Bito H. Tsien R.W. Neuron. 1996; 16: 89-101Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, 33Bito H. Deisserroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar, 47Barthel F. Boutillier A.L. Trousland J. Loeffler J.P. Neuroscience. 1996; 70: 1053-1065Crossref PubMed Scopus (16) Google Scholar). Our EMSA, which uses core promoter sequence of C/EBPβ gene as a probe, suggests that CREB can control the expression of C/EBPβ in hippocampal neurons (Fig. 3). Given that the expression of C/EBPβ can be controlled by CREB, C/EBPβ activity is predicted to be enhanced by Ca2+ signals in hippocampal neurons. To examine this possibility, hippocampal neurons in cultures were subjected to membrane depolarization by incubating them with high K+ solution for 5 s (33Bito H. Deisserroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (977) Google Scholar). Four hours after membrane depolarization, nuclear extracts were prepared from both depolarized neurons and control neurons incubated with normal Tyrode's solution. EMSA using a C/EBP binding site as a probe showed that the DNA binding activity of C/EBPβ is augmented by membrane depolarization of hippocampal neurons (Fig. 6 A). The DNA binding activity of C/EBPδ is also found to be enhanced by membrane depolarization (Fig. 6 A). The increase of C/EBPβ mRNA was observed in hippocampal neurons 4 h after membrane depolarization, suggest
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