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MCNMolecular and Cellular Neuroscience 14, 455–467 (1999)

Article ID mcne.1999.0804, available online at http://www.idealibrary.com on

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euronal Depolarization Enhances theranscription of the Neuronal Serine Protease

nhibitor Neuroserpin

hilipp Berger, Serguei V. Kozlov, Paolo Cinelli, Stefan R. Kruger,

orenz Vogt, and Peter Sonderegger1

nstitute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland

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euroserpin is an axonally secreted neuronal serine proteasenhibitor. Based on its inhibitory activity towards tissue plas-

inogen activator (tPA) and its predominant expression in theerebral cortex, the hippocampus, and the amygdala, a roleor neuroserpin in the regulation of neural plasticity has beenuggested. We recently found that neuroserpin mRNA isncreased in cultured hippocampal neurons upon depolariza-ion with elevated extracellular KCl. Using luciferase reporteronstructs containing segments of the promoter region ofhe neuroserpin gene, we identified a 200-bp segment near theranscription initiation site that is responsible for both theeuron-specific expression of the neuroserpin gene and thenhanced transcription resulting from depolarization. Nerverowth factor, which alone had no effect on the expression ofeuroserpin mRNA in hippocampal neurons, had a markedotentiating effect when supplied in combination with ele-ated extracellular KCl. In contrast, the transcription factorif/268 blocked neuroserpin transcription. These results impli-ate neuroserpin as an activity-regulated modulator of tPActivity at the synapse and provide further support for theccurrence of activity-regulated proteolytic processes at theynapse.

NTRODUCTION

Neural plasticity, the ability to undergo adaptivetructural and functional changes, is one of the crucialequirements for the functional integrity of the nervousystem of vertebrates. It is generally accepted thatlectrical activity, or particular patterns of electricalctivity, plays a crucial role in the induction of neurallasticity by triggering molecular and structural changes

1

sm

To whom correspondence should be addressed at the Institute ofiochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057urich, Switzerland. Fax: 141–1–635 68 31. E-mail: pson@bioc.unizh.ch.

044-7431/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

esulting in altered synaptic functions as well as reorga-ization in neural circuits (Katz and Shatz, 1996). A vastariety of cellular or molecular mechanisms has been

mplicated in the induction or the implementation oflastic changes (Chen and Tonegawa, 1997). In the pastears, a number of studies have revealed evidence of aole for tissue plasminogen activator (tPA) in neurallasticity. tPA is upregulated as an immediate-earlyene upon induction of long-term potentiation (LTP) inhe rat hippocampus (Qian et al., 1993) and in cerebellarurkinje cells after complex motor learning tasks (Seedst al., 1995). Correspondingly, hippocampal LTP waserturbed in mice lacking the tPA gene (Frey et al., 1996;uang et al., 1996). In a recent study using hippocampal

ultures, it was shown that secreted tPA contributes toxon elongation and the formation of new presynapticaricosities. These changes can be blocked by the appli-ation of inhibitors of tPA (Baranes et al., 1998). Theolecular mechanism involving tPA in the context of

TP has not been determined so far. However, based ontudies of the role of tPA in excitotoxicity-mediatedeuronal cell death resulting from kainate-induced hip-ocampal seizures, a proteolytic cascade comprising

PA and plasmin was postulated (Tsirka et al., 1995).aminin was identified as the first target of the tPA/lasmin cascade in the nervous system. Therefore, the

ncreased tPA activity observed after a seizure in theippocampus contributes to neuronal death by degrad-

ng the molecular scaffold on which the neurons arenchored (Chen and Strickland, 1997). It is conceivablehat the mechanism resulting in neuronal cell death afterxcessive electrical stimulation subserves a fine-tunedynaptic remodeling under more physiological stimuli

uch as those resulting in hippocampal LTP or cerebellarotor learning.

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We have recently demonstrated that the neuronalerine protease inhibitor neuroserpin is a potent inhibi-or of tPA and also inhibits plasmin (Krueger et al., 1997;sterwalder et al., 1998). In situ studies of developing

nd adult mice (Krueger et al., 1997) revealed the highestevel of neuroserpin mRNA expression in the neocortex,he hippocampal formation, the olfactory bulb, and themygdala, but no mRNA was found in the caudateutamen or the thalamic nuclei. In the cerebellum, theranule cells and a subset of the Purkinje cells were positiveor neuroserpin. This expression pattern, grossly overlap-ing with that of tPA (Thewke and Seeds, 1996; Qian et al.,993), implicates neuroserpin as a regulator of tPA in theNS. In the present study, we demonstrate that neuroserpinRNA is upregulated in cultured hippocampal neurons by

epolarization and report on the cis-acting sequences andhe transcription factors involved in the enhancement andeduction of neuroserpin gene expression.

ESULTS

levated Extracellular KCl Increases NeuroserpinRNA in Primary Cultures of Hippocampal Neurons

Previous studies on tissue sections revealed thateuroserpin mRNA is mainly expressed in neurons of

he developing and adult mouse brain (Krueger et al.,997). To address the question whether a neuron-pecific expression of neuroserpin is also maintained inissociated primary neural cultures, hippocampal cellsere cultivated according to Banker and Cowan (1977).sing in situ hybridization, we found neuroserpinRNA exclusively in cells exhibiting a clear neuronalorphology (Fig. 1A). In contrast, tPA is expressed in

euronal and some nonneuronal cells (Fig. 1B). For theuantification of these two mRNAs, dissociated hippo-ampal cells from E17.5 mouse embryos were culturedor 10 to 12 days to allow the formation of a densexo-neuritic network rich in synaptic connections. There-fter, the cells were exposed to an elevated concentra-ion of extracellular KCl (55 mM) for different timeeriods. The mRNAs of neuroserpin and tPA wereetermined by quantitative RT-PCR using a mutated

1) strand RNA as an internal control. As demonstratedn Fig. 1E, an increase in the neuroserpin mRNA from.10 to 0.22 pg/µg total RNA was found after an 8-hxposure to elevated KCl, and a further increase to 0.32g/µg total RNA was observed if the exposure tolevated KCl was extended to 40 h. In contrast, themount of neuroserpin mRNA was not increased in the

arly phase of the depolarization. Parallel measure-ents of the mRNA for tPA, the presumed cognate

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arget protease of neuroserpin in the CNS, confirmedhe nature of tPA as an immediate-early gene activatedy medium change (Fig. 1F). As previously describedConanan and Crutchley, 1983), the change of mediumesulted in a transient increase of tPA mRNA in the cellsith normal and elevated KCl. No statistically signifi-

ant difference between the two conditions was found.owever, after 40 h, when a pronounced increase of

euroserpin mRNA was found in cells exposed tolevated KCl, the tPA mRNA in cells exposed to aormal KCl concentration was reduced to the starting

evel, whereas in the cells exposed to elevated KCl, tPARNA was decreased to approximately 50%.

euroserpin Gene Promoter Fragments Driveell-Type-Specific Expression of Luciferaseeporters in PC12 Cells

Four different cell lines, PC12, HeLa, 3T3, and COS,ere transfected with a series of luciferase reporter

onstructs containing fragments of the neuroserpinromoter reaching 70 bp into exon 1 and extending into

he 58 flanking region by, respectively, 24, 78, 125, 346,11, 1153, 2724, and 3941 bp (Fig. 2A; for the location ofhe transcription start site see Berger et al., 1998). Aonstruct with the SV40 promoter in front of the lucifer-se cDNA served as a control. To normalize the data, theells were cotransfected with a plasmid containing thescherichia coli b-galactosidase gene under the control of

he human cytomegalovirus promoter. The transfectionfficiency was good in PC12, COS, and HeLa cells, butoor in 3T3 cells. As demonstrated in Fig. 2B, theeuroserpin gene promoter exhibited highest activity in

he neural crest-derived cell line PC12 (Greene andischler, 1976). In the nonneuronal cell lines tested, onlyconsiderably lower (HeLa cells) or no (COS and 3T3

ells) activity was observed. The SV40 control promoterxhibited a similar activity in all tested cell lines.

In the PC12 cells, the highest activities were associ-ted with the longest constructs, namely pGL3–3941bp,panning from nucleotide 23941 to 170; pGL3–2724bp,panning from nucleotide 22724 to 170; and pGL3–153bp, spanning from nucleotide 21153 to 170. Step-ise shortening of the longest promoter segment in-

luded in the reporter construct to 2724, 1153, 511, 346,nd 125 bp did not result in a significant reduction of theranscriptional activity, although some variation wasbserved. The construct including the first 125 bp of the8 flanking region and the first 70 bp of exon 1,GL3–125bp, exhibited nearly the same activity as the

ongest construct. Further shortening of the promoteregment, in contrast, resulted in a marked decrease of

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Transcriptional Regulation of Neuroserpin mRNA 457

he transcriptional activity to approximately 50% withGL3–78bp, and only minimal, if any, activity wasbserved with the shortest construct, pGL3–24bp. Thetepwise loss of activity of the pGL3–78bp and theGL3–24bp construct suggested that the region between125 and 224 contains at least two cis-acting positive

IG. 1. Expression of neuroserpin and tPA in hippocampal cultures. (euroserpin and a tPA antisense riboprobe. (C and D) Hybridizationeuroserpin and tPA mRNA in depolarized hippocampal cultures byith NGF and KCl. Total RNA was prepared from the cells and the amsing a mutated RNA as internal standard. The values on the y axis are

egulatory elements important for the promoter activity.igure 3A shows a summary of potential binding sites

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or transcription factors in this region as describedefore (Berger et al., 1998). If the region between 224nd 170 was omitted from the constructs, the activityas clearly reduced (Figs. 3B and 3D), indicating that

he correct transcription initiation site is important forhe promoter activity. Constructs corresponding in their

d B) In situ hybridization of a low-density hippocampal culture with aa neuroserpin and a tPA sense probe. (E and F) Quantification of theR. Hippocampal cells were cultured for 12 days prior to stimulationof neuroserpin and tPA mRNA was determined by quantitative PCR

n in pg/µg total RNA. They represent means 6 SD (n 5 6).

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8 extensions, but bearing in addition parts of intron 1nd exon 2 (see Fig. 6A), showed the same activity

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attern, but the level of expression was twofold higherdata not shown).

In HeLa cells the corresponding activities were threeo four times lower. Maximal activities were achieved

ith the pGL3–1153bp and the pGL3–125bp constructs.nclusion of additional segments from more upstreamegions (21153 to 23941) did not result in enhancedctivity. Again, the pGL3–78bp construct retained 50%f the maximal activity (Fig. 2B).

euroserpin Gene Promoter Fragments Areesponsive to Cell Depolarization and aombination of Cell Depolarization and NGF

IG. 2. Characterization of the neuroserpin gene promoter by luciferausing a 150-bp blunt-ended AvaI fragment to the luciferase cDNA. Airectly adjacent segment of the 58 flanking region. The 58 end of the se

he transcription start site (for the experimental determination see Bergeporter constructs and the luciferase activity was normalized to theMV-lacZ. For each cell line, the activity of the plasmid pGL3-Basic cbserved in the neural crest-derived PC12 cells, whereas only little (Hata represent means 6 SD obtained from triplicates. Each experiment

PC12 cells respond to the addition of NGF with ahange in the pattern of gene expression that results in a

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orphological differentiation into neuron-like cellsGreene and Tischler, 1976). Moreover, elevated levels of

1 ions in the extracellular medium induces membraneepolarization and increases the membrane conduc-

ance for Na1 and Ca21 ions (DeLorme and McGee,988). Therefore, to test whether NGF and/or elevatedotassium concentrations elicited a change in the expres-ion of the neuroserpin gene, transfected PC12 cellsere subjected to the two stimuli (30 ng/ml NGF and 55M KCl) separately or together. Depolarizing the cellsith elevated KCl resulted in a markedly enhanced

xpression with most constructs (Fig. 3B). A smallncrease was already found with the pGL3–78bp con-

porter studies. (A) The luciferase reporter constructs were obtained bystructs contained 70 bp of exon 1, the transcription start site, and thet contained in the different constructs is marked by the distance froml., 1998). (B) PC12, HeLa, COS, and 3T3 cells were transfected with thelactosidase activity derived from the cotransfected control plasmid

ining the SV40 promoter was defined as 100%. Maximal activity wasor no (COS, 3T3) activity was observed in nonneuronal cell lines. Thecarried out at least twice.

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Transcriptional Regulation of Neuroserpin mRNA 459

ere necessary. NGF alone did not influence the pro-oter activity, but it strongly potentiated the response

o elevated KCl. Together, NGF and KCl increased theromoter activity 2.5- to 3.5-fold. Whereas no potentiat-

ng effect of NGF was observed for the constructGL3–78bp, the expression of the pGL3–125bp con-

IG. 3. Induction of neuroserpin expression in PC12 cells by depolescribed in Berger et al. (1998). (B) Transfected PC12 cells were cultur5 mM KCl. Note that the minimal construct is not stimulated. (C) Cohe cells and does not depend on osmolarity. Cultures were treated witr 200 µM APV and 10 µM nifedipine. The SV40 promoter is influonstructs containing nucleotides 2125 to 224 or 278 to 224, indicatihe pGL3-Basic vector was used as a control and is not affected bybolished the synergistic effect of NGF. The data represent means 6 S*P # 0.01, **no significant difference; Student’s t test).

truct was clearly enhanced by NGF, suggesting thep1/AP2 site located from position 274 through 283 as

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possible region responsible for the potentiating actionf NGF.As demonstrated in Figs. 3B and 3C, PC12 cells

ransfected with the luciferase cDNA under the controlf the SV40 promoter or with the minimal constructGL3–24bp or the pGL3-Basic vector did not show any

tion and NGF. (A) Potential binding sites for transcription factors as40 h in 10% FCS/DMEM supplemented with 30 ng/ml NGF and/or

assays to show that the induction is due to an influx of Ca21 ions intoF/55 mM NaCl or NGF/KCl alone or together with 40 µM verapamil

by neither NGF/KCl nor NGF/NaCl. (D) Luciferase assays withat this region contains at least two elements responsible for induction.lation. (E) Mutation of the CAAT box in the pGL3–125bp constructained from triplicates. Each experiment was carried out at least twice

arizaed forntrolh NGencedng th

ranscriptional enhancement after stimulation with NGFnd KCl. Likewise, exposure to NGF and NaCl instead

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f NGF and KCl did not increase luciferase expression,xcluding an osmotic effect as explanation for thenhanced activity (Fig. 3C). Several antagonists of cal-ium channels were tested for their ability to abolish theostimulation by NGF and KCl. A complete blockageas obtained with nifedipine, an inhibitor of L-type

oltage-dependent calcium channels. Verapamil, an-ther known blocker of L-type voltage-dependent cal-ium channels, also reduced the promoter activity signifi-antly (Fig. 3C). The transcription-stimulating effect ofepolarization could not be reversed by blocking of theodium channel with tetrodotoxin (data not shown).his result is in line with the recently reported observa-

ion that the addition of KCl to the medium acts directlyia voltage-dependent calcium channels (Higuchi et al.,996). Taken together, these results indicate that annflux of calcium ions into the cell is necessary to induceeuroserpin gene expression.To further narrow down the region with the depolariza-

ion-inducible cis-acting elements, luciferase constructs lack-ng the region from 224 to 170 were used. As demon-trated in Fig. 3D, the DNA fragment ranging from 224 to125 retained the full inducibility upon depolarization.ecause the smaller fragment from 224 to 278 exhibitednly half of the inducibility, two separate elements, one

ocated between 278 and 2125 and the other between 224nd 278, are likely to contribute to the transcriptionaltimulation that occurs upon depolarization.

Because the CAAT box from nucleotide 259 throughucleotide 266 (see Berger et al., 1998) represented anbvious candidate for a cis-acting element in this region, weutated the CAAT box in the construct pGL3–125bp. Using

he same luciferase reporter assay under conditions oflevated extracellular KCl as well as a combination oflevated KCl and NGF (Fig. 3E), we found that the depolar-zation-induced transcriptional activation also occurs in thebsence of a functional CAAT box. However, with both theonstruct containing the mutation-inactivated CAAT boxnd the construct containing the CAAT box but lacking therea upstream of position 278, the potentiating effect ofGF was lacking. These results suggest a complex mecha-ism of depolarization-induced transcriptional activationnd its potentiation by NGF, in which elements upstreamnd downstream of position 278 take part.

ootprint and Band-Shift Analyses Identifyranscription Factor Binding Sites near theranscription Initiation Site of theeuroserpin Gene

The region flanking the transcription initiation site ofhe neuroserpin gene has recently been characterized

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Berger et al., 1998). In close vicinity to the transcriptiontart site, the 58 upstream region of the gene contains anp1 site, a CAAT box, and a region with a combinedp1/AP2 site (Fig. 3A). To examine the functionality ofhese binding sites, a genomic fragment spanning nucle-tides 2124 to 1113 with respect to the transcription

nitiation site was analyzed by DNase I footprintingFig. 4). Several protected areas became visible whenuclear extracts from PC12 (uninduced and inducedith KCl/NGF), HeLa, and COS cells were used. The

egions most prominently protected spanned nucleotide25 to 253, nucleotide 256 to 271, and nucleotide 271

o 291. Based on the published sequence of the neuroser-in gene promoter region (Berger et al., 1998), the

ootprint observed between nucleotides 253 and 225ncludes an Sp1 binding site. This segment was pro-ected with all types of nuclear extracts. The CAAT boxocated at 256 to 271 was clearly protected with HeLaell extract and to a considerable degree with extractsrom PC12 cells, but not with extracts from 3T3 (data nothown) and COS cells. The third protected region (271o 297) overlapped with a combined AP-2 and Sp1inding site and became visible with all tested extracts.n additional protected region was observed betweenosition 2310 and 2289. This region was equallyrotected with extracts from PC12, COS, and 3T3 cells. Itontains potential binding sites for AP-2 and Sp1 (dataot shown).For the quantification and identification of the factorhich binds to the protected CAAT box, gel retardation

xperiments were performed with an oligonucleotideuplex spanning the proximal Sp1 site and the CAATox and using extracts from induced and uninducedC12, COS, and HeLa cells. Three shifted complexes

C1–C3) with different mobilities and intensities werebserved (Fig. 5A). All three complexes disappearedhen the assay was carried out in the presence of an

xcess of unlabeled oligonucleotide. The most promi-ent complex, C2, was present in all three cell lines

ested, the strongest signal being found with the extractf HeLa cells. With COS cell extract, 20 times moreuclear proteins were necessary to obtain a C2 complexith a similar yield. The CAAT box is a known recogni-

ion sequence for the transcription factor C/EBPb/LAP,hich is expressed in PC12 cells (Fawcett et al., 1996).herefore, a supershift assay with anti-LAP antibodies

kindly provided by Dr. U. Schibler, Geneva) wasarried out, and an interference with the formation ofhe C2 complex was observed (data not shown). Toromote the blockage of the C/EBPb transcription

actor prior to the initiation of the experiment, theuclear extract from PC12 cells was preincubated with

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Transcriptional Regulation of Neuroserpin mRNA 461

he anti-LAP antibodies. As demonstrated in Fig. 5B, theormation of the C2 complex was prevented by annti-C/EBPb, but not by a control (anti-NrCAM) anti-ody, indicating that the transcription factor C/EBPbinds to the CAAT box in PC12 cells.

otransfection Experiments Identify zif/268 as ailencer of the Neuroserpin Gene

Because of the reported role of zif/268 as an immedi-

IG. 4. DNase I footprint analysis of the basal promoter of the neurxtract from HeLa, COS, unstimulated PC12, and NGF/KCl-stimulateranscription factors are marked on the left side; positions relativ

axam-Gilbert C 1 T sequencing reaction; w/o, without nuclear extra

te-early gene induced by various forms of neuronallectrical activity, the zif/268 site located at the 58 end of

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ntron 1 (Fig. 3A, Berger et al., 1998) was investigatedith regard to a transcription-regulatory function. PC12

ells were cotransfected with luciferase reporter geneonstructs and with zif/268 expression constructs in theSCT-1 vector. In addition to the constitutive overexpres-ion of zif/268 under the CMV promoter, we alsoxpressed an antisense mRNA to block the endogenousxpression of zif/268 in PC12 cells (Bartel et al., 1989;ata not shown). As a control, the insertless pSCT-1ector was used. Cotransfection of the zif/268 antisense

in gene. Footprinting analysis was performed with 50 µg of nuclear2 cells. The protected regions and the corresponding binding sites for

the transcription initiation site are given on the right site. (C1T,

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onstruct and a series of intron-containing promoteronstructs (Int; Fig. 6A) which contain a potential

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462 Berger et al.

if/268 binding site just after exon 1, doubled theuciferase activity. The activities of the intronless con-tructs (Sta) and that of the pGL3-Basic vector with theV40 promoter were not affected by any of the zif/268xpression vectors (Fig. 6B). Therefore, we concludedhat the endogenously expressed zif/268 might act as ailencer of the transcription of the neuroserpin pro-oter.

ISCUSSION

In the present study we found that the expression ofhe gene encoding the axonally secreted neuronal serinerotease inhibitor neuroserpin is transcriptionally upreg-lated by cell depolarization with elevated extracellularCl. Addition of NGF, which alone had no effect oneuroserpin gene transcription, resulted in a markedotentiation of the effect observed with KCl alone. This

IG. 5. Band-shift analysis of the basal promoter of the neuroserpin gox and the Sp1 site and nuclear extracts from PC12 (1 µg), NGF/Komplexes (C1–C3) with different mobilities and intensities were obsligonucleotide. (B) The formation of the C2 complex can be blockednti-NrCAM serum does not block the complex formation. ns, nonspe

bservation implicates neuroserpin as a regulatory ele-ent in activity-dependent synaptic processes.

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Neuroserpin was originally identified as an axonallyecreted protein of cultured chicken embryonic dorsaloot ganglia neurons (Stoeckli et al., 1989) and subse-uently cloned in chicken (Osterwalder et al., 1996),ouse (Krueger et al., 1997), and human (Schrimpf et al.,

997). Recent results revealed that tPA and to a lesserxtent uPA and plasmin, but not thrombin, can benhibited in vitro by the recombinant neuroserpin pro-ein (Osterwalder et al., 1998). In an extensive in situybridization study neuroserpin was found to be ex-ressed by neurons in various areas of the CNS as wells the PNS (Krueger et al., 1997). The selective regionalattern of expression in the adult mouse brain sug-ested a tissue-specific transcriptional control of theeuroserpin gene. Because the strongest expression ofeuroserpin in the adult CNS was found in neurons ofighly ‘‘plastic’’ structures, such as the cerebral cortex,

he hippocampus, and the amygdala, we tested whethereuroserpin mRNA was changed by electrical activity.

(A) Gel retardation assay with an oligonucleotide spanning the CAATated PC12 (1 µg), HeLa (1 µg), and COS (20 µg) cells. Three shifted. All three complexes can be competed with an excess of unlabeled

eincubation of the nuclear PC12 extract with an anti C/EBPb serum.band; F, free probe.

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sing quantitative PCR, we found that cultured hippo-ampal neurons depolarized by elevated KCl contained

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Transcriptional Regulation of Neuroserpin mRNA 463

ignificantly higher amounts of neuroserpin mRNA.herefore, we set out to study the transcriptional regula-

ion of the neuroserpin gene. Reporter gene, footprint-ng, and gel retardation assays were performed toharacterize the neuroserpin promoter. We found that ahort segment of only 200 bp of the promoter region wasufficient to drive transcription of the gene in PC12 cells.nly a minor activity of the reporter constructs was

ound in HeLa cells, whereas the construct was com-letely inactive in COS and 3T3 cells. By depolarizing

he PC12 cells, a clearly enhanced promoter activity wasnduced. NGF alone did not enhance the promoterctivity, but exerted a strong potentiating effect inCl-stimulated cells. Further studies with PC12 cells

evealed that the induction depends on an influx ofalcium ions into the cells and that the combinedp1/AP-2 site and the CAAT box are responsible for thectivation of the neuroserpin promoter. Several of the

IG. 6. Cotransfection experiments with pSCT zif/268. (A) Sche-atic representation of the cloning procedure for the intron-

ontaining constructs which contain the endogenous zif/268 bindingite placed after exon 1. The cloning of the standard constructs lackinghe zif/268 binding site is described in Fig. 1A. (B) PC12 cells wereransfected with the luciferase reporter gene constructs and differentorms of the pSCT eukaryotic expression vector. Cotransfection withhe pSCT antisense-zif/268 vector increased the activity twofold in thease of the intron-containing constructs (Int). The intronless constructsSta) and pGL3-Basic vector with the SV40 promoter were not affectedy any of the expression vectors. pSCT zif/268, sense; pSCT anti-zif/68, antisense; pSCT-1, empty vector. The data represent means 6 SDbtained from triplicates. Each experiment was carried out at leastwice.

AAT-binding transcription factors have been impli-ated in the upregulation of gene transcription after

at

nflux of calcium ions into the cell (Roy et al., 1996; Akirat al., 1990). For example, in the activation of the C3omplement gene in the acute phase of infection (for aeview, see Volanakis, 1995), a switch from C/EBPa to/EBPd is observed after stimulation of the cells with

nterleukin-1 (Juan et al.,1993). In PC12 cells we foundhat the CAAT box of the neuroserpin promoter isccupied by C/EBPb. C/EBPb, but not C/EBPa, isnown to participate in the activation of promoters in aissue-specific manner by forming stable heterodimers

ith Sp1 (Lee et al., 1997). In the basal neuroserpinromoter the C/EBPb binding site is flanked by twoinding sites for Sp1. Therefore, two different het-rodimers might be formed. As a speculation, the distalp1 factor could form a heterodimer with C/EBPb,hereby contributing to the transcription of the neuroser-in gene. On the proximal site, Sp1 could form aeterodimer with an Inr-binding protein, which is impor-ant for the binding of the basal transcription machineryLee et al., 1993). Recent studies demonstrated an impor-ant role for C/EPBb in the consolidation of long-term

emory. ApC/EBP, the homologue of C/EBPb in Aply-ia, is upregulated as an immediate early gene byerotonin (Alberini et al., 1994). Because the DNA bind-ng affinity of C/EPBb is enhanced by phosphorylationy MAPK, it is conceivable that the potentiating actionf NGF is mediated via an activation of MAPK by NGFMarshall, 1995; Martin et al., 1997).

The neuroserpin promoter contains a potential bind-ng site for zif/268 in the first intron close to exon 1 atosition 1103 (for details see Berger et al., 1998). The

nfluence of different zif/268 expression vectors washerefore tested with the intron-containing constructs,

hereas the intronless constructs without the zif/268ite were used as controls. To our surprise we found thathe basal transcription of zif/268 in PC12 cells wasufficient to suppress the activity of the neuroserpinromoter. The expression of a zif/268 antisense RNAesulted in a twofold increase in the activity. Althougheveral target genes are known to be upregulated byif/268, inhibition of transcription by zif/268 has beeneported before. For example, binding of zif/268 to theromoter of the adenosine desaminase gene suppresses

ranscriptional activity (Ackerman et al., 1991). In thisase the inhibitory action of zif/268 was shown to beediated by competition with an Sp1 site. Because in

he neuroserpin promoter no Sp1 site is found overlap-ing with or in the vicinity of the zif/268 site, theownregulation of the neuroserpin gene transcriptionust be mediated by another mechanism. Conceivable

re an interaction of zif/268 with the binding of theranscription initiation machinery and a preliminary

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ermination of the transcription by transcription elonga-ion block. In vitro and in vivo, the expression of themmediate-early gene zif/268 is highest 4 h after anxternal induction and decreases to background levelsithin 24 h (Christy et al., 1988; Cole et al., 1989). The

ranscription factor zif/268 could therefore function toold down the expression of neuroserpin in the earlyesponse after an external stimulus. A role for zif/268 inelaying the upregulation of neuroserpin is supportedy the observation that PC12 and primary neuronal cellultures produce both tPA and zif/268 as immediate-arly genes (Milbrandt, 1987; Gualandris et al., 1996),hereas neuroserpin expression is delayed. Togetherith the published observations, the results presented

ere support the notion that zif/268 could play a role inoordinating the concerted expression of tPA and itsognate inhibitor, neuroserpin, and thus enable both theunction of tPA as an immediate-early gene after activity-ependent transcription and the subsequent termina-

ion of tPA function by neuroserpin.The activity-regulated transcription of the neuroser-

in gene is in line with the observations made on theole of tPA in learning and memory operations and inippocampal LTP (Frey et al., 1996; Baranes et al., 1998).y its activity-dependent transcriptional activation inombination with a zif/268-mediated suppression, neu-oserpin might play an important modulatory role inhe control of tPA activity at synapses, at the level ofoth single neurons and multicellular neuronal circuits.

XPERIMENTAL METHODS

n Situ Hybridization of Hippocampal Cultures

Hippocampi were dissected from E17.5 embryonicice, dissociated, and plated on poly-D-lysine (Sigma)

t a density of 105 cells per 3.5-cm cell culture dish. Theultures were maintained under serum-free conditionsn DMEM with 2 mM sodium pyruvate and B27 supple-

ent (Gibco). After 6 days in culture, the cells were fixedith 4% paraformaldehyde in PBS. The cells were then

ncubated for 10 min in 0.25% acetic anhydride in 0.1 Mriethanolamine, permeabilized for 10 min with 0.2 M

Cl, washed with 43 SSC, and prehybridized in hybrid-zation buffer (53 SSC, 53 Denhardt’s solution, 250g/ml total yeast RNA, and 500 µg/ml herring spermNA). For hybridization, the cells were incubated over-ight at 55°C with hybridization buffer containing 300g/ml of a DIG-labeled mouse neuroserpin or tPA

robe (sense or antisense) (Krueger et al., 1997). Thelates were then subjected to low-stringency (23 SSC at

t5

oom temperature) and high-stringency (0.23 SSC at5°C) washes. Hybridized probe was detected using anlkaline phosphatase-coupled anti-digoxigenin anti-ody (Boehringer) and the substrates nitroblue tetra-olium blue and x-phosphate (Boehringer).

uantitative RT-PCR from Hippocampal Cultures

Hippocampal neurons were dissected from E17.5mbryonic mice, dissociated, and plated on poly-D-ysine (Sigma) at a density of 106 cells per 3.5-cm cellulture dish in DMEM complemented with 10% FCS.oth glial and neuronal cell types are present under

hese culture conditions. After 10 to 12 days in culture,he cells were exposed to 55 mM KCl and 30 ng/ml NGFnd harvested after 1.5, 8, and 40 h. Total RNA fromippocampal cultures was prepared with the QiagenNAeasy total RNA kit and quantified photometrically.o increase the accuracy and reproducibility of theT-PCR, a mutated, in vitro-transcribed control RNAas added to the RNA of the hippocampal cultures.The cDNAs from mouse neuroserpin and tPA, both in

he Bluescript vector pBS KS(1), were partially digestedith EcoRI, filled in with Klenow polymerase, and

eligated. The new vectors, pBS mNSmut and pBSPAmut, lost their EcoRI restriction sites at position 464nd position 804, respectively, but acquired newly gen-rated AseI restriction sites at these positions. ControlNA for quantification was obtained by in vitro transcrip-

ion from the mutated plasmids according to standardrocedures (Sambrook et al., 1989) and the DNA tem-late was subsequently removed with DNase I (Rocheiagnostics). Reverse transcription was performed in a

0-µl reaction volume containing 0.5 µg total RNA, 0.1o 10 pg control RNA, 1 mM each dNTP, 50 mM Tris–HCl,H 8.3, 70 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 2.5M random hexamers, 1 unit RNasin (Promega), and 150nits MuLV reverse transcriptase (Promega). Samples were

ncubated for 5 min at room temperature, 30 min at 42°C,nd 10 min at 60°C and heat inactivated at 95°C prior to theCR. The following primers were used for PCR: mNS-quant-

or (58-GCG ATG GGA ATG ATG GAG C-38), mNS-quant-ack (58-CTC CGG AGA CAC CAG ATC-38), tPA-quant-for58-CGG AGT TCT GTA GCA CAC C-38), and tPA-quant-ack (58-TCC TTC ATC ACA TGG CAC C-38). The PCReaction was performed in a 20-µl reaction volume contain-ng 8 µl of the RT reaction, 0.2 µM each forward andackward primer, 4 mM (for neuroserpin) or 2 mM (for tPA)gCl2, 0.2 mM each dNTP, and 1 unit of Taq DNApolymer-

se (Promega) on a PTC-200 cycler (MJ Research, Water-

own, MA) for 40 cycles with an annealing temperature of5°C. The PCR fragments were cut with EcoRI or AseI,

s(I

R

cofl(wB7cpfN(SwpTtCT(ptKcIp

tEtvltCmtNm2w1rsa

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(Lbpbrvhnbv

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Transcriptional Regulation of Neuroserpin mRNA 465

eparated on a 3% agarose gel, stained with SYBR Green 1Molecular Probes, Eugene, OR), and quantified on a Fluor-mager 575 (Molecular Dynamics, Sunnyvale, CA).

eporter Gene Assays

Different parts of the putative promoter region wereloned into the pGL3-Basic vector containing the cDNAf the firefly luciferase as a reporter gene (Promega). Touse a DNA fragment of the neuroserpin gene with theuciferase cDNA, a 150-bp blunt-ended AvaI fragment172 to 278 relative to the transcription initiation site)as cloned into the blunt-ended BglII/HindIII-cut pGL3-asic vector to produce the precursor construct pGL3–8bp. All other plasmids of this series were obtained byloning more upstream regions of the promoter into theGL3–78bp vector using the HindIII site (224) and the

ollowing endogenous restriction sites: KpnI (2125),coI (2347), BssHII (2511), HindIII (21135), BglII

21410), PstI (22088), SacI (22724), and XbaI (23941).ites with no counterparts in the pGL3-Basic polylinkerere blunt ended with either Klenow or T4 DNAolymerase and fused to the SmaI site of the polylinker.he CAAT box was mutated using a PCR strategy with

he oligonucleotides MutCat (58-GAG CTG AAG CTTTT CTG AGA TTC AAG TCC GCC TTC CCC GCCCT AAA CTT ACG AAG CTC G-38) and pGL3sense

58-ATA GTA CTA ACA TAC GC-38) with the vectorsGL3–78bp and pGL3–125bp as templates. The ob-

ained fragments and the template vector were cut withpnI and HindIII and religated. The identity of allonstructs was confirmed by DNA sequencing with theRD41-labeled pGL3sense primer or the pGL3backrimer (58-AGC CTT ATG CAG TTG C-38).DNA for transfection experiments was purified with

he Qiagen EndoFree system or over a CsCl gradient.ach construct has been tested in triplicate with at least

wo different DNA preparations. The pGL3-promoterector containing the SV40 promoter and the promoter-

ess pGL3-Basic vector were used as positive and nega-ive controls in each transfection experiment. PC12, 3T3,OS, and HeLa cells were grown in DMEM supple-ented with 10% FCS. Studies of transcription-regula-

ory effects were performed by addition of 30 ng/mlGF, 55 mM KCl, 10 µM nifedipine, or 40 µM verapa-il. Cells were preplated 4 h prior to transfection in

4-well plates (1.5 cm2/well) at a density of 40%. Theyere washed twice with PBS and then incubated with

50 µl OPTIMEM 1 (Gibco BRL) containing 0.15 pmoleporter gene construct, 0.75 µg CMV-lacZ as an internal

tandard, pGL3-Basic to a total amount of 1.75 µg DNA,nd 7 µg Lipofectin. After 3 h, the transfection solution

[m

as removed and the cells were incubated in mediumupplemented with 100 U/ml penicillin/0.1 mg/mltreptomycin (Gibco BRL) and the substances used fornduction. Calcium channel antagonists were added 1 hrior to induction. Cells were collected after 40 h in 200l reporter lysis buffer (Promega) and centrifuged brieflynd the supernatant was frozen at 270°C for at least 1 h.

The luciferase activity was monitored in a luminometerMBV Biocounter, Vevey, Switzerland) by mixing 50 µluciferase Assay Reagent (Promega) and 5 µl cell lysate.-Galactosidase activity was determined in a microtiterlate containing 30 µl cell lysate, 1 mM MgCl2, 45 mM-mercaptoethanol, 1 mg/ml o-nitrophenyl-b-D-galactopy-anoside, and 100 mM sodium phosphate, pH 7.5, in a totalolume of 150 µl. The absorption was measured after 3 to 4in a Bio-Rad Model 2550 ELISA reader at 405 nm. For

ormalization, the luciferase activity was divided by the-galactosidase activity. The activity of the pGL3-Promoterector was defined as 100% for each cell line.

reparation of Nuclear Extracts

Nuclear extracts were prepared according to a protocol ofillimann and Trueb (1994). In brief, 2 3 108 cells wereashed twice with ice-cold PBS, collected in buffer A2 (10M Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM

GTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyluoride, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 10 µg/mlprotinin, 10 µg/ml antipain), gently vortexed, and incu-ated on ice for 15 min. To isolate the nuclei, 10% Triton-100 was added to a final concentration of 0.75% and theamples were vortexed for 10 s and put on ice for 5 min.uclei were pelleted by a 2-min centrifugation at 14,000g

nd nuclear proteins were extracted with 175 µl buffer C220 mM Hepes, pH 7.9, 400 mM NaCl, 25% glycerol, 0.1 mMDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and protease

nhibitors as above). Nuclear debris were removed byentrifugation and the supernatants were frozen in liquiditrogen. Protein concentrations were determined with theio-Rad protein assay using BSA as a standard.

ootprinting Assay

A 285-bp EcoRI–BssHII fragment containing the ge-omic sequence from 2124 to 1113 was excised from

he plasmid pBluescript II KS(1) NarI–KpnI/237 andurified with QiaExII. Two hundred nanograms of the

ragment was unidirectionally labeled at the BssHII siteith [32P]dCTP in a 30-µl reaction containing 10 mM

ris–HCl, pH 7.9; 10 mM MgCl2; 50 mM NaCl; 1 mMithiothreitol; 150 µM dATP, dGTP, and dTTP; 30 µCi

a-32P]dCTP; and 5 units Klenow polymerase. After 30in at room temperature, the reaction was stopped and

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B

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B

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466 Berger et al.

he nucleotides were removed with the Qiagen nucleo-ide removal kit. The binding was performed in a totalolume of 25 µl by incubation of 50 to 75 µg nuclearxtract protein, 1 µg poly(dI–dC), and 100,000 cpmabeled fragment in incubation buffer (15 mM Tris–HCl,H 7.9, 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, 1M dithiothreitol, 20% glycerol) for 10 min on ice

ollowed by 10 min at room temperature. LimitedNase digestion was achieved by addition of 2 vol ofNase buffer (10 mM MgCl2, 5 mM CaCl2) and 0.25 to

.0 units of DNase I (Roche Diagnostics). Reactions wereerminated after 150 s at room temperature with 100 µltop buffer (200 mM NaCl, 20 mM EDTA, 1% SDS, 50g/ml yeast tRNA), extracted twice with phenol/hloroform/isoamyl alcohol (25/24/1) and once withhloroform, and then precipitated with ethanol. Theellet was resuspended in 10 µl loading dye, denaturedt 85°C for 5 min, and loaded on a 6% polyacrylamide/rea/TBE gel. Maxam–Gilbert G1A sequencing ladders

Sambrook et al., 1989) and fragments digested in thebsence of nuclear extract were used as references.

el Retardation Assay

To form a double-stranded DNA fragment spanninghe region from 272 to 224, two complementary oligo-ucleotides (BS-for, 58-CGA GCT TCG GCC AAT TAGGG CGG GGA AGG CGG ACT TGA ATC TCA-38;S-back, 58-CTT CTG AGA TTC AAG TCC GCC TTCCC GCC TCT AAT TGG CCG A-38) were synthesized

Microsynth AG, Balgach, Switzerland). Both oligo-ucleotides (2.5 µmol of each) were heated for 5 min in00 mM NaCl at 95°C and then slowly cooled to 4°C.verhanging 58 ends were filled-in with Klenow poly-erase in a reaction containing 10 pmol double-

tranded oligonucleotide; 50 mM Tris–HCl, pH 7.9; 100M NaCl; 10 mM MgCl2; 1 mM dithiothreitol; 200 µM

ach dCTP, dGTP, and dTTP; 20 µCi of [a-32P]dATP; andunits of Klenow polymerase. The fragments were

urified with the QIAquick nucleotide removal kit toemove unincorporated nucleotides.

A typical binding reaction contained 50,000 cpm ofabeled oligonucleotide (approx 0.1 pmol) and 1 to 20 µgf nuclear extracts in 10 mM Hepes, pH 7.9, 80 mM KCl,mM EDTA, 1 mM EGTA, 2 µM ZnCl2, 0.01% Triton-100, and 12% glycerol. The reaction mixture was

ncubated for a further 20 min at room temperature,ollowed by a 10-min incubation at 4°C. For competitionssays, the nuclear extracts were preincubated over-ight at 4°C with 1 µl antiserum, followed by a 5-min

ncubation with the radiolabeled probe. Samples were

oaded without loading dye on a 5% native polyacryl-mide gel (0.53 TBE) and electrophoresed for 3 h at 4°C.

C

otransfection Experiments with zif/268

A second series with progressive deletions of the 58anking region contained in addition parts of intron 1 and

he noncoding region of exon 2, fused via the endogenousranslation start codon to the luciferase cDNA (Fig. 6A). Forxpression of mouse zif/268, the 58 overhangs of thevaII-cut cDNA (ATCC, No. 63027) were blunt-ended withlenow polymerase and cloned into the EcoRV/PvuII-cutukaryotic expression vector pSCT-1 (Pedrocci et al., 1994).he resulting vectors were controlled for the orientation of

he insert and named pSCT zif/268 and pSCT antisense-zif/68. They contained the full coding region under the controlf the CMV promoter in sense or antisense direction,orrespondingly. Cotransfection experiments were per-ormed with 0.5 µg CMV-lacZ, 1.0 µg pGL3 reporter vector,nd 1.0 µg of pSCT zif/268, pSCT antisense-zif/268, orriginal, insertless pSCT-1 vector.

CKNOWLEDGMENTS

We thank Dr. U. Schibler for providing anti-LAP antibodies, Dr. O.eorgiev for helpful discussions, and Dr. Ned Mantei for critically

eading the manuscript. This research was supported by the Swissational Science Foundation, the Helmut Horten Foundation, thelga Mayenfisch Foundation, the Jublilaumsstiftung der Rentenans-

alt/Swisslife, and the Novartis Foundation.

EFERENCES

ckerman, S. L., Minden, A. G., Williams, G. T., Bobonis, C., andYeung, C. Y. (1991). Functional significance of an overlappingconsensus binding motif for Sp1 and Zif268 in the murine adenosinedeaminase gene promoter. Proc. Natl. Acad. Sci. USA 88: 7523–7527.kira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y.,Nakajima, T., Hirano, T., and Kishimoto, T. (1990). A nuclear factorfor IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBOJ. 9: 1897–1906.lberini, C. M., Ghirardi, M., Metz, R., and Kandel, E. R. (1994).C/EBP is an immediate-early gene required for the consolidation oflong-term facilitation in Aplysia. Cell 76: 1099–1114.

anker, G. A., and Cowan, W. M. (1977). Rat hippocampal neurons indispersed cell culture. Brain Res. 126: 397–442.

aranes, D., Lederfein, D., Huang, Y. Y., Chen, M., Bailey, C. H., andKandel, E. R. (1998). Tissue plasminogen activator contributes to thelate phase of LTP and to synaptic growth in the hippocampal mossyfiber pathway. Neuron 21: 813–825.

artel, D. P., Sheng, M., Lau, L. F., and Greenberg, M. E. (1989). Growthfactors and membrane depolarization activate distinct programs ofearly response gene expression: Dissociation of fos and jun induc-tion. Genes Dev. 3: 304–313.

erger, P., Kozlov, S. V., Krueger, S. R., and Sonderegger, P. (1998).Structure of the mouse gene for the serine protease inhibitorneuroserpin (PI12). Gene 214: 25–33.

hen, C., and Tonegawa, S. (1997). Molecular genetic analysis of synapticplasticity, activity-dependent neural development, learning, and memoryin the mammalian brain. Annu. Rev. Neurosci. 20: 157–184.

C

C

C

C

D

F

F

G

G

H

H

J

K

K

K

L

L

M

M

M

O

O

P

Q

R

S

S

S

S

T

T

V

Transcriptional Regulation of Neuroserpin mRNA 467

hen, Z. L., and Strickland, S. (1997). Neuronal death in the hippocam-pus is promoted by plasmin-catalyzed degradation of laminin. Cell91: 917–925.

hristy, B. A., Lau, L. F., and Nathans, D. (1988). A gene activated inmouse 3T3 cells by serum growth factors encodes a protein with‘‘zinc finger’’ sequences. Proc. Natl. Acad. Sci. USA 85: 7857–7861.

ole, A. J., Saffen, D. W., Baraban, J. M., and Worley, P. F. (1989). Rapidincrease of an immediate early gene messenger RNA in hippocampalneurons by synaptic NMDA receptor activation. Nature 340: 474–476.

onanan, L. B., and Crutchley, D. J. (1983). Serum-dependent induc-tion of plasminogen activator in human fibroblasts by catechol-amines and comparison with the effects of prostaglandin E1.Biochim. Biophys. Acta 759: 146–153.eLorme., E. M., and McGee, R., Jr. (1988). Effects of prolongeddepolarization on the nicotinic acetylcholine receptors of PC12 cells.J. Neurochem. 50: 1248–1252.

awcett, T. W., Eastman, H. B., Martindale, J. L., and Holbrook, N. J.(1996). Physical and functional association between GADD153 andCCAAT/enhancer-binding protein beta during cellular stress. J.Biol. Chem. 271: 14285–14289.

rey, U., Muller, M., and Kuhl, D. (1996). A different form oflong-lasting potentiation revealed in tissue plasminogen activatormutant mice. J. Neurosci. 16: 2057–2063.reene, L. A., and Tischler, A. S. (1976). Establishment of a noradrener-gic clonal line of rat pheochromocytoma cells which respond tonerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424–2428.ualandris, A., Jones, T. E., Strickland, S., and Tsirka, S. E. (1996).Membrane depolarization induces calcium-dependent secretion oftissue plasminogen activator. J. Neurosci. 16: 2220–2225.iguchi, H., Nakano, K., Kim, C. H., Li, B. S., Kuo, C. H., Taira, E., andMiki, N. (1996). Ca21/calmodulin-dependent transcriptional activa-tion of neuropeptide Y gene induced by membrane depolarization:Determination of Ca(21)- and cyclic AMP/phorbol 12-myristate13-acetate-responsive elements. J. Neurochem. 66: 1802–1809.uang, Y. Y., Bach, M. E., Lipp, H. P., Zhuo, M., Wolfer, D. P., Hawkins,R. D., Schoonjans, L., Kandel, E. R., Godfraind, J. M., Mulligan, R.,Collen, D., and Carmeliet, P. (1996). Mice lacking the gene encodingtissue-type plasminogen activator show a selective interferencewith late-phase long-term potentiation in both Schaffer collateraland mossy fiber pathways. Proc. Natl. Acad. Sci. USA 93: 8699–8704.

uan, T. S., Wilson, D. R., Wilde, M. D., and Darlington, G. J. (1993).Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.Proc. Natl. Acad. Sci. USA 90: 2584–2588.

atz, L. C., and Shatz, C. J. (1996). Synaptic activity and the construc-tion of cortical circuits. Science 274: 1133–1138.

raner, S. D., Chong, J. A., Tsay, H. J., and Mandel, G. (1992). Silencingthe type II sodium channel gene: A model for neural-specific generegulation. Neuron 9: 37–44.

rueger, S. R., Ghisu, G. P., Cinelli, P., Gschwend, T. P., Osterwalder, T.,Wolfer, D. P., and Sonderegger, P. (1997). Expression of neuroserpin,an inhibitor of tissue plasminogen activator, in the developing andadult nervous system of the mouse. J. Neurosci. 17: 8984–8996.

ee, J. S., Galvin, K. M., and Shi, Y. (1993). Evidence for a physical

interaction between the zinc-finger transcription factors YY1 andSp1. Proc. Natl. Acad. Sci. USA 90: 6145–6149.

W

Johnson, P. F. (1997). The ability of C/EBP beta but not C/EBP alphato synergize with an Sp1 protein is specified by the leucine zipperand activation domain. Mol. Cell. Biol. 17: 2038–20347.arshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling:Transient versus sustained extracellular signal-regulated kinaseactivation. Cell 80: 179–185.artin, K. C., Michael, D., Rose, J. C., Barad, M., Casadio, A., Zhu,H. X., and Kandel, E. R. (1997). Map kinase translocates into thenucleus of the presynaptic cell and is required for long-termfacilitation in Aplysia. Neuron 18: 899–912.ilbrandt, J. (1987). A nerve growth factor-induced gene encodes apossible transcriptional regulatory factor. Science 238: 797–799.sterwalder, T., Contartese, J., Stoeckli, E. T., Kuhn, T. B., andSonderegger, P. (1996). Neuroserpin, an axonally secreted serineprotease inhibitor. EMBO J. 15: 2944–2953.sterwalder, T., Cinelli, P., Baici, A., Pennella, A., Krueger, S. R., Schrimpf,S. P., Meins, M., and Sonderegger, P. (1998). The axonally secreted serineprotease inhibitor, neuroserpin, inhibits plasminogen activators andplasmin but not thrombin. J. Biol. Chem. 273: 2312–2321.

edrocchi, M., Schafer, B. W., Mueller, H., Eppenberger, U., andHeizmann, C. W. (1994). Expression of Ca(21)-binding proteins ofthe S100 family in malignant human breast-cancer cell lines andbiopsy samples. Int. J. Cancer 57: 684–690.ian, Z., Gilbert, M. E., Colicos, M. A., Kandel, E. R., and Kuhl, D.(1993). Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation.Nature 361: 453–457.

oy, B., Li, W. W., and Lee, A. S. (1996). Calcium-sensitive transcrip-tional activation of the proximal CCAAT regulatory element of thegrp78/BiP promoter by the human nuclear factor CBF/NF-Y. J. Biol.Chem. 271: 28995–29002.

ambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: ALaboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.

chrimpf, S. P., Bleiker, A. J., Brecevic, L., Kozlov, S. V., Berger, P.,Osterwalder, T., Krueger, S. R., Schinzel, A., and Sonderegger, P.(1997). Human neuroserpin (PI12)—cDNA cloning and chromo-somal localization to 3q26. Genomics 40: 55–62.

eeds, N. W., Williams, B. L., and Bickford, P. C. (1995). Tissueplasminogen activator induction in Purkinje neurons after cerebel-lar motor learning. Science 270: 1992–1994.

toeckli, E. T., Lemkin, P. F., Kuhn, T. B., Ruegg, M. A., Heller, M., andSonderegger, P. (1989). Identification of proteins secreted from axons ofembryonic dorsal-root-ganglia neurons. Eur. J. Biochem. 180: 249–258.

hewke, D. P., and Seeds, N. W. (1996). Expression of hepatocytegrowth factor/scatter factor, its receptor, c-met, and tissue-typeplasminogen activator during development of the murine olfactorysystem. J. Neurosci. 16: 6933–6944.

sirka, S. E., Gualandris, A., Amaral, D. G., and Strickland, S. (1995).Excitotoxin-induced neuronal degeneration and seizure are medi-ated by tissue plasminogen activator. Nature 377: 340–344.

olanakis, J. E. (1995). Transcriptional regulation of complementgenes. Annu. Rev. Immunol. 13: 277–305.

illimann, T. E., and Trueb, B. (1994). Identification of functionalelements and reconstitution of the alpha 1(VI) collagen promoter. J.

ee, Y. H., Williams, S. C., Baer, M., Sterneck, E., Gonzalez, F. J., and Biol. Chem. 269: 332–338.

Received August 11, 1999Revised September 27, 1999

Accepted September 27, 1999