Proc. Natl. Acad. Sci. USAVol. 90, pp. 7054-7058, August 1993Immunology

Interleukin 1 induction of the c-jun promoterK. MUEGGE*, M. VILA*, G. L. GUSELLA*, T. Musso*, P. HERRLICHt, B. STEINt, AND S. K. DURUM**Biological Carcinogenesis and Development Program, Program Resources Inc./Dyncorp, National Cancer Institute-Frederick Cancer Research andDevelopment Center and Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21702-1201; andtKernforschungszentrum, Institut fuer Genetik and Toxikologie, Karlsruhe, Federal Republic of Germany

Communicated by Paul B. Beeson, April 26, 1993

ABSTRACT Interleukin 1 (IL-1) induces pleiotropic ef-fects in many cell types during inflammation and immunity. Wehave recently shown how the IL-1 signal is transmitted to thenudeus: In T cells and in pituitary cells, IL-1 induced genes viaactivation ofthe nuclear factor AP-1. We now demonstrate howIL-1 activates the AP-1 factor in liver cells, which are a majortarget for IL-1 during the acute phase response in vivo. IL-1induced gene transcription ofboth AP-1 components, c-jun andc-fos. IL-1 also increased the stability of c-jun mRNA. Wedefine two enhancer sites in thejun promoter that are requiredfor induction by IL-1. Although the binding sites share somesimilarity with the AP-1 binding site, the nuclear factorsbinding thejun motifs are not composed ofJun or Fos proteins.Thus these data identify two binding proteins that serve as oneof the rtst nuclear targets for IL-1 signal transduction.

Interleukin 1 (IL-1) is a major regulator of inflammation andimmunity (1, 2). Although two IL-1 receptors have beenrecently cloned (3, 4), little is understood about the intracel-lular mechanism of IL-1 signal transduction in different celltypes. We have recently demonstrated that IL-1 induces IL-2gene transcription in a T-cell line via activation of thetranscription factor AP-1 (5, 6). Furthermore, IL-1 enhances,B-endorphin secretion in a pituitary cell line via AP-1 (7). TheAP-1 motif is a 9-bp-long conserved sequence originallydefined in phorbol ester [phorbol 12-myristate 13-acetate(PMA)]-inducible promoters (8, 9). IL-1 also activates someof these PMA-inducible promoters, including those of thecollagenase gene (10, 11), the metallothionein gene (12), andthe simian virus 40 promoter (K.M. and S.K.D., unpub-lished). The AP-1 sequence binds either a homodimer of thec-jun gene product or a heterodimer formed by c-jun and c-fosproducts (reviewed in ref. 13). Products of other members ofthe jun and fos gene families can also participate in AP-1complex formation, although complexes formed with thejun-B product have been reported to inhibit, rather thanpromote, transcription (14).

In a T cell, we found that IL-1 required a costimulus forAP-1 activation as well as for IL-2 gene transcription (5, 6).The costimulus is provided by T-cell receptor triggering. Thistwo-signal requirement is explained by IL-1 inducing anincrease of c-jun mRNA, which is only one component of theAP-1 factor, whereas T-cell receptor triggering induced thec-fos component (5, 6).The liver responds to IL-1 with induction of a number of

genes, some of which carry a functional AP-1 site (e.g., themetallothionein gene). We therefore examined hepatocytesas a third target cell for IL-1 effects. Here we report that, incontrast to T cells, IL-1 alone is a sufficient signal for AP-1activation in hepatocytes and requires no costimulus foractivation of both c-jun and c-fos components. We alsodescribe the mechanism of c-jun induction and we further-

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more identify the nuclear factors involved in activation of thec-jun promoter.

MATERIALS AND METHODSCell Culture. The human hepatoma cell line HepG2

(ATCC) was maintained in Dulbecco's modified Eagle'smedium supplemented with 10o fetal calf serum, 100 ,uMnonessential amino acids, 1 mM sodium pyruvate, 2 mMglutamine, 100 units of penicillin per ml, and 100 jig ofstreptomycin per ml. Before IL-1 stimulation, the fetal calfserum concentration was reduced to 0.5% for 12 hr (or 6 hrbefore transfection).

Transfection. The cells were transiently transfected usingthe DEAE method as described (15). After transfection thecells were incubated for 24 hr and then stimulated for afurther 24-hr period. Chloramphenicol acetyltransferase(CAT) activity was measured in cell lysates as described (16)and is expressed as percent acetylation. Data are the mean ofat least three experiments in which CAT enzyme activity wasnormalized according to the amount of plasmid uptake asmeasured by blot analysis (17). The AP-1-CAT constructcontained three collagenase AP-1 sites linked to the thymi-dine kinase promoter and the CAT gene (9) (this construct isalso called 3xTRE-tk-CAT, since the AP-1 site has beenformerly called IPA responsive element). AP-1-CAT wascompared to a control tk-CAT, which was the same constructbut lacking the AP-1 sites (9). Thejun plasmids consisted of1600 bp upstream or 283 bp upstream and 170 bp downstreamof the transcriptional start site of the c-jun promoter linked tothe CAT gene; these plasmids were mutated in two "AP-1-like" sites to determine the role of these sites in c-juninduction by UV radiation, phorbol ester (18), and IL-1 asdescribed here. One of these AP-1-like sites was previouslyimplicated in positive feedback of AP-1 on production of itsJun component (19). In the point-mutated forms of thejun-CAT construct the AP-1-like motifs were exchanged: inmutant 1 sequence -71 TGACATCAT -63 is replaced by-71 ATCCACCAT -63 and sequence -191 ATTACCT-CATC -181 is replaced by -191 TGGACCTCGAG -181(18).Northern Blot Analysis. Cells were stimulated with the

indicated agents for 30 min. To inhibit transcription, cellswere preincubated for 20 min with 10 pg ofactinomycinD perml, which inhibited 90% of the overall RNA synthesis (mea-sured by [3H]uridine incorporation). Total cytoplasmic RNAwas extracted (20) and 15 pg was fractionated on 1% agarosegel. Blots were hybridized with 32P-labeled cDNA probes forhuman c-fos and v-jun or mouse 18S genes.

Transcriptional Run-On Analysis. Cells were stimulatedwith the indicated agents for 15 min, nuclei were extracted,and run-on analysis was performed as described (21). Briefly,200 id of frozen nuclei suspension was thawed, mixed with200 ,ul of reaction buffer {10 mM Tris HCl, pH 8.0/5 mM

Abbreviations: IL, interleukin; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase.

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MgCl2/300 mM KCl/0.5 mM ATP, CTP, and GTP/100 ,Ciof [at32P]UTP (1 Ci = 37 GBq)}, and incubated for 30 min at30°C. Newly synthesized 32P-labeled RNA was isolated andhybridized at 42°C for 36 hr to DNA immobilized on Nytran.The immobilized cDNA probes included human c-fos, mouse18 S, a 100-mer oligonucleotide with the human c-jun se-quence, and pBR322 (as a negative control).

Gel Retardation Assay. Cells were washed of completeculture medium and then cultured in medium containing 0.5%fetal calfserum for 12 hr. Cells were then stimulated with IL-1for 20 min and nuclear extracts were prepared as described(22). The assay was performed by incubation of 2-5 ,ul ofnuclear extract for 20 min at room temperature in 25 ,ul ofreaction buffer containing 2 &g poly(dI-dC), 10% glycerol, 25mM Hepes (pH 7.6), 1 mM dithiothreitol, 50 mM NaCl,0.05% Nonidet P-40, and 20,000 cpm of 32P end-labeleddouble-stranded oligonucleotide. Complexes were separatedon 6% polyacrylamide gels as described (23). For competi-tion, 10-50 ng of unlabeled oligonucleotide was added 5 minprior to addition of the labeled oligonucleotide. A concen-tration of 25 ng equals a 100-fold molar excess. To determineif nuclear factors contained Jun or Fos proteins, 5 ,ul ofnuclear extract was preincubated for 2 hr at 4°C with 2 ul ofspecific rabbit antiserum against v-jun or v-fos (a gift ofH. J.Rahmsdorf) or normal rabbit serum as indicated. The oligo-nucleotides used had the following sequence: junl, -77 TTGGGG TGA CAT CAT GGG CTA -57;jun2, -197 CGG AGCATT ACC TCA TCC CGT -177; collagenase, -79 TAAAGC ATG AGT CAG ACA CCT CTG -56; NFKB of theimmunoglobulin K chain enhancer, -3958 CAG AGG GGACTT TCC GAG AGG C -3937.

RESULTSIL-1 Activates the AP-1 Factor in Human Hepatoma Cells.

IL-1 activates the transcription of several genes containingAP-1 sites, including the collagenase (10, 11) and metallothio-nein genes (12). In the collagenase gene, the AP-1 site hasbeen shown to be essential for activation of the promoter inhuman hepatoma cells (11, 15, 24). We therefore directlytested whether IL-1 could activate AP-1 in the human hep-atoma line HepG2. mRNA for the type I IL-1 receptor (3) isexpressed in this cell line (K.M. and S.K.D., unpublished).As shown in Table 1, IL-1 induced a 3-fold increase in activityfrom an AP-1-CAT construct (9) consisting of three AP-1motifs from the collagenase promoter. Therefore IL-1 aloneis a sufficient signal in HepG2 cells to activate the AP-1factor, whereas in our earlier study in T cells (5, 6), IL-1 alonewas not sufficient but acted as a cofactor, together with asignal from the T-cell antigen receptor.

Previous studies indicated that induction of the transcrip-tion factor AP-1 is partly posttranslationally and partly tran-

Table 1. IL-1 activates the c-jun promoter via AP-1-like sites

% acetylation Fold

Without With inductionConstruct IL-1 IL-1 by IL-1

(-1600/+170)jun-CAT 6.2 15 2.6 ± 0.4Mutant 1 (71-63) 1.3 1.2 <1.0Mutant 2 (190-183) 1.9 1.5 <1.0Double mutant (1 + 2) 0.4 0.5 <1.1AP-1-CAT 7.7 23.6 3.0 ± 0.2

HepG2 cells were transiently transfected with 10 pg of the geneconstructs as indicated. After 24 hr cells were washed in phosphate-buffered saline and then stimulated with IL-1,8 (294 pM) or leftuntreated. CAT activity was analyzed in cell lysates 24 hr later. Dataare expressed as percentage of substrate acetylated, normalized foramount of transfected DNA for each construct within each experi-ment, and are the mean values of three independent experiments.

scriptionally regulated (18). Since new protein synthesis isrequired for some of the IL-i-induced genes-for example,the collagenase (24) and stromelysin (25) genes-it waspossible that this represented synthesis of the AP-1 compo-nents. The AP-1 factor that binds to the collagenase motifconsists of at least two members of the jun andfos protoon-cogene families (13). As shown in Fig. la, the mRNAs forboth components, c-jun and c-fos, were increased by IL-1after 30 min ofstimulation. The induction ofc-jun mRNA was12- to 15-fold above background. PMA, used as a positivecontrol, induced both protooncogene mRNAs to a similarmagnitude (data not shown). The expression of c-jun andc-fos mRNA was transient, peaking at 30 min and decreasingto <20%/o at 60 min after stimulation with IL-1 (data notshown). Thus IL-1 induced increases in mRNAs for bothcomponents ofAP-1 in HepG2, in contrast to T cells in whichIL-1 only induced an increase in the c-jun component,whereas antigen receptor induced the c-fos component. Thisis consistent with IL-1 alone inducing AP-1 activity inHepG2, compared to IL-1 plus antigen receptor inducingAP-1 in T cells.To determine whether the increase in c-jun and c-fos

mRNA was due to increased transcription, HepG2 cells wereincubated with the transcriptional inhibitor actinomycin Dprior to stimulation. Actinomycin D blocked >90% of totalRNA synthesis (measured by [3H]uridine incubation, datanot shown), and, as shown in Fig. la, no mRNA increase

a Northern Analysis

c-jun

c-fos 0

18S 9

0-J -n

_~ _+ z

El--

b Nuclear Run On Analysis

_il l. 18S

Control

c-los

..# c-jun(-) ILI

c mRNA Stability100 \

\

501

- + IL-1

IL

30 45Time, min

FIG. 1. IL-1 induces c-jun and c-fos mRNA via increased tran-scription and mRNA stability. (a) HepG2 cells were stimulated for 30min as indicated with human recombinant IL-1p3 at a concentrationof 294 pM. Cells were incubated with the transcriptional inhibitoractinomycin D (Act D; 10 .g/ml) for 30 min and then stimulated withIL-1 for another 30 min. Total cytoplasmic RNA was extracted andprobed with cDNA probes for 32P-labeled v-jun or human c-fos ormouse 18 S. (b) HepG2 cells were stimulated for 15 min as indicatedwith human recombinant IL-1,8. Nuclei were extracted and in vitrotranscription was performed for 30 min at 30°C. The 32P-labeledRNAwas isolated and hybridized to immobilized DNA probes for c-jun,c-fos, 18 S as a positive control, and pBR322 as a negative control.The experiment shown is representative of two independent exper-iments performed. (c) HepG2 cells were stimulated for 30 min withPMA (30 ng/ml). The intensity of c-jun mRNA detected at this timepoint was set as 100%. After stimulation with PMA, cells wereincubated with actinomycin D for 20 min and then for another 10 or25 min in the presence (o) or absence (o) of 100 units of humanrecombinant IL-113 per ml. Total cytoplasmic RNA was extracted atvarious times and analyzed for c-jun mRNA. Three independentexperiments were performed, showing 1.5-, 3.5-, and 7-fold differ-ences of IL-1 over control at 30 min.

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occurred after actinomycin D treatment. Therefore transcrip-tion is necessary for IL-1 induction of c-jun and c-fos mRNA.To determine whether IL-1 directly induced transcription

of the AP-1 components, nuclear run-on analysis was per-formed. HepG2 nuclei were extracted 15 min after stimula-tion with IL-1 (since c-jun mRNA peaked at 30 min). Asshown in Fig. lb, IL-1 induced c-jun as well as c-fos genetranscription -5-fold above controls without IL-1. PMA,used as a positive control, activated the c-jun promoter to asimilar magnitude as IL-1 (data not shown). Thus the genesfor both components of the AP-1 factor, c-jun and c-fos, aretranscriptionally induced by IL-1.The increase in c-jun mRNA was 12- to 15-fold above

control; however, transcriptional regulation only accountedfor =5-fold enhancement. We therefore considered that theremay be additional posttranscriptional control of the c-jungene by IL-1 to account for the difference. Thus we analyzedmRNA stability. The c-jun mRNA was first induced by PMA,and then further transcription was blocked by the use ofactinomycin D. IL-1 was then added and the amount of c-junmRNA was analyzed at various time points. Shown in Fig. lcis one representative experiment. Within 30 min, IL-1 stabi-lized c-jun mRNA, increasing its half-life from 8-14 min to20-25 min. On the other hand, c-fos mRNA stability was notsignificantly enhanced by IL-1 (data not shown). Thus IL-1can regulate the c-jun component of the AP-1 factor throughtranscriptional and posttranscriptional mechanisms.

IL-1 Activates the c-jun Promoter via junl and jun2 BindingSites. Having shown that IL-1 activated c-jun transcription, wethen analyzed the c-jun promoter to define the IL-i-responsiveelements. The c-jun promoter region has been recently cloned.The cloned genomic fragment consists of 1600 bp upstreamand 170 bp downstream ofthe transcriptional start site (18, 19).The c-jun promoterwas linked to the CATgene and transientlytransfected into HepG2 cells. Thejun-CAT construct showeda high background activity probably because either the trans-fection procedure or serum components activated the c-junpromoter. Nevertheless, IL-1 induced the c-jun promoter 2- to3-fold above controls in the absence of IL-1 (Table 1). Otherreports (18, 19) have indicated a similar range of inducibilityafter stimulation with PMA. A shorterjun promoter construct(-283/+170) showed no reduction of CAT activity (data notshown) and indicated that the essential enhancer elementswere located downstream of -283.Two functional sites have been recently described in the

c-jun promoter as analyzed by DNase I footprinting and theuse of point-mutated jun-CAT constructs in transient trans-fections (18, 19). Both enhancers, junl and jun2, are locatedat -71 and -191 upstream ofthe transcriptional start site. Wetested whether these sites were involved in c-jun induction byIL-1. Mutants of the c-jun promoter (18) that were pointmutated in the junl or jun2 motif or at both were transientlytransfected into HepG2 cells. As shown in Table 1, mutationof either jun motif abrogated inducibility by IL-1 and de-creased basal expression of the promoter. Mutation of bothjun sites reduced IL-i-induced CAT activity, as well as basalexpression, to <7% compared to the full-length promoter.The low expression of the mutants was due to a true lack ofresponse, not simply a low uptake of DNA during transfec-tion (this was shown by extracting DNA from transfectedcells, titrating, and blotting with a CAT probe, which indi-cated equal DNA uptake). Both jun sites are thereforerequired in the c-jun promoter for induction by IL-1 as wellas for basal activity. Activation of the jun promoter occurswithin 15 min (Fig. lb) and protein synthesis is not requiredfor c-jun mRNA induction by IL-1, since cycloheximide doesnot inhibit IL-1 induction of this message (data not shown).This suggests that IL-1 posttranslationally activates pre-formed nuclear factors binding to the junl and the jun2 motifin the jun promoter and induces gene transcription.

Nuclear Factors Binding to the c-jun Promoter: MotifsSimilar to AP-1 but Proteins Serologically Distinct from Junand Fos. Both enhancer motifs, junl and jun2, containsequences homologous to the AP-1 binding site. Moreover, ithad been reported that overexpression ofc-jun could enhancec-jun transcription in HeLa cells (19). We therefore testedwhether AP-1 factors are the endogenous transcription fac-tors in HepG2 cells that bind to the junl and jun2 sites.To characterize the nuclear factors binding to the two jun

motifs, we performed gel retardation assays with IL-1-induced nuclear extracts of HepG2 cells. Nuclear proteinswere detected that bound to thejunl andjun2 motifs (Fig. 2),and this binding was specific, as shown by competition withthe unlabeled oligonucleotide but not with the unrelatedNFKB motif (Fig. 3). The mutation in jun2 that reducedpromoter function (shown in Table 1) also reduced nuclearprotein binding by 80% as detected in the gel retardationassay (not shown). Two specific complexes binding to eitherjunl orjun2 were dominant (Figs. 2 and 3) and differed fromthe complex formed by the AP-1 factor binding to the AP-1site of the collagenase promoter. To determine whether oneof thejun binding complexes (e.g., the slower migrating one)could consist of an AP-1 factor we used purified AP-1 factorfrom HeLa cells. Fig. 2b shows that the purified AP-1 factorbinds to the collagenase AP-1 motif but not to junl or jun2sequences. We have also noted that the bacterially expressedc-Jun and v-Fos complex binds with a 7-fold lower affinity tothejunl motif than to the collagenase motif (data not shown).These findings suggest that it was unlikely that the complexesbinding junl or jun2 are bona fide AP-1 factors, a point thatwill be verified serologically (below).Although the proteins binding junl and jun2 motifs were

probably not typical AP-1 factors, these motifs have never-theless some similarity to the collagenase motif. We nextdetermined whether they were similar enough to competewith one another for binding to their respective nuclearproteins (Fig. 3). This was the case, in that the binding ofHepG2 nuclear extracts to junl, jun2, or the collagenase

a Hep G2

.j

X,.3'.a

b Purified AP-1

Q ._

FIG. 2. Differences in binding specificity of purified AP-1 versusHepG2 extracts. (a) HepG2 cells were stimulated with IL-1 (294 pM).Nuclear extracts were incubated for 20 min with radioactive end-labeled oligonucleotides consisting of either the collagenase (Coil.),junl, orjun2 motifs and separated by acrylamide gel electrophoresis.(b) Purified AP-1 factor from HeLa cells was incubated for 20 minwith either the collagenase, junl, or jun2 motif and separated byacrylamide gel electrophoresis.

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a Collagenase motif b junl motif

c jun2 motif

4.4,,k

I

N _ - xc e O Lz

I a -a C.) z

en U)

z F O

c c -U.

.'. ._. o z

FIG. 3. junl and jun2 are members of the AP-1 family by thecriterion of cross-competition. (a) IL-1-induced nuclear extracts ofHepG2 cells were incubated with a radioactive labeled collagenase(Coil.) AP-1 motif. Competitors used were 50 ng of either theunlabeledjunl, jun2, the collagenase AP-1 motif, or the NFKB motif.(b) IL-1-induced nuclear extracts ofHepG2 cells were incubated withthejunl motifand competition was performed as indicated. (c) Sameas b using the jun2 motif.

motif was competitively inhibited successfully by all threemotifs, although at high competitor concentrations (200-foldexcess). However, the competition was best when the unla-beled oligonucleotide matched the labeled oligonucleotide.For example, competition for binding to the jun2 motif washighest forjun2 itself and decreased in the order jun2 > junl> AP-1 motif (titration analysis not shown). This againsuggested that the factors binding to the jun motifs weredistinct from a typical AP-1 complex.

Typical AP-1 factors are dimers of c-Jun and c-Fos;however, either c-Jun or c-Fos is capable of dimerizationwith several other proteins containing leucine zippers, andc-Jun can form homodimers. We therefore examined whetherthe proteins binding junl or jun2 contained either c-Jun orc-Fos components. Fig. 4 shows that specific antiserumagainst Jun (recognizing the products of the jun familyincluding v-jun, c-jun, junB, and junD) or against v-Fos didnot inhibit proteins that bound to either junl or jun2. More-over, thejunl orjun2 complexes did not show a supershift (aretardation caused by the bound antibody), which shouldhave occurred if an AP-1 factor were part of the jun com-plexes but not involved in DNA binding. As a positivecontrol, these antisera inhibited binding of typical AP-1factors to the collagenase AP-1 motif as shown (Fig. 4a). Thisindicates that the typical c-Jun/c-Fos AP-1 factors werepresent in these nuclear extracts and that these antiseracompletely inhibited their DNA binding, but that these AP-1factors are distinct from the proteins that bind junl or jun2,which contained neither c-Jun nor c-Fos components.

DISCUSSIONWe have recently demonstrated that IL-1 activates the IL-2gene in T cells (5, 6) and f-endorphin secretion in pituitary

Uz cF n

z.,F t

z d

FIG. 4. Nuclear factors binding tojunl andjun2 do not react withantibodies against Jun or Fos. (a) IL-1-induced nuclear extracts ofHepG2 cells were preincubated with specific antisera against Jun andFos. Gel retardation assay was then performed with a radioactivelabeled probe of the collagenase motif. (b) Same as a using the junlprobe. (c) Same as a using thejun2 probe. NRS, normal rabbit serum.

cells (7) via the transcriptional factor AP-1. Other nuclearfactors reported to be induced by IL-1 (reviewed in ref. 26)are NFKB (27) and NF-IL6 (28). Because the liver is animportant IL-1 target, we here analyzed the human hepatomacell line HepG2 for AP-1 activation. We show that IL-1activates the c-Jun/c-Fos AP-1 factor in this cell type usingmultiple mechanisms of control. In the first 15 min after IL-1engages its receptor, preformed transcription factors becomeactivated. These factors bind to motifs in the jun promoterthat are similar to AP-1 motifs, but the factors are distinctfrom the Jun/Fos AP-1 factor. Besides transcriptional regu-lation, IL-1 enhances the amount of c-jun mRNA furtherthrough increased stability. Since transcription ofc-fos is alsoinduced by IL-1, the resulting increase in Jun and Foscomprises the activated AP-1 factor, which, in turn, controlsmany genes.We found that the activation of c-jun transcription by IL-1

is controlled by two sites, junl at -71 and jun2 at -191.During the course of these studies, these sites have also beenidentified as responsive elements to several other stimuli,including phorbol ester, UV light, okadaic acid, or theoncogene v-mos (18, 29, 30-32). An additional site at position-59 has recently been identified that controls c-jun inductionin response to epidermal growth factor (33).The two c-jun promoter sites show some sequence simi-

larity with the AP-1 binding site of the collagenase promoter,but nevertheless the factor that they bind is distinct from anAP-1 factor by the following criteria: (i) there are multiplecomplexes binding tojunl orjun2 that are distinct in size fromthe bonafide AP-1 factor binding to the collagenase gene, (ii)the purified AP-1 factor binds with much lower affinity to thejun sites than to a true AP-1 site, (iii) cross-competitionsshow differences in the affinity of the endogenous nuclearproteins forjun versus AP-1 motifs, and (iv) specific antiserado not recognize either Jun or Fos components in the jun

a Collagenase motif

...p...

b. junl motif

3*

c jun2 motif

I

CC C ILI Q .. .. Z

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complexes. Furthermore, the adenoviral oncogene ElA,which inhibits the Jun/Fos complex, did not inhibit activationof thejun promoter (34), also suggesting that nuclear factorsother than c-Jun and c-Fos bind to the jun promoter.

Since the jun promoter is activated within 15 min and denovo protein synthesis is not required, IL-1 probably post-translationally activates the junl and jun2 binding factors.This activation of junl and jun2 binding factors is notreflected in increased DNA binding but presumably occursby increased transcriptional activity, as is also observed forsome of the leucine zipper proteins such as CREB andC/EBP,3. Phosphorylation is a common mechanism for ac-tivation of transcription factors (35). Positions 63 and 73 ofthe Jun protein require for transcriptional activation phos-phorylation, which can be performed by microtubule-associated protein 2 (MAP-2) kinase (36). Most recentlyMAP-2 kinase has been implicated in signal transduction byIL-1 (37). Thus MAP-2 kinase could serve as a link betweenIL-1 receptor binding and gene activation not only for AP-1activation but also for posttranscriptional regulation of thejunl and jun2 proteins that induce the c-jun promoter.The intracellular pathway leading from IL-1 through AP-1,

part of which is described in the present paper, is probablyalso used by other extracellular signals since AP-1 is nowbelieved to mediate many such growth and differentiationsignals. Thus the junl and jun2 binding proteins may be theearliest transcription factors in many cellular responses.

We thank J. J. Oppenheim and D. Longo for their suggestions onthe manuscript, T. Bos and P. Vogt for providing the v-jun probe, M.Karin for purified AP-1 factor, and H. J. Rahmsdorf for specificantiserum against v-jun and v-fos. This project has been funded atleast in part with federal funds from the Department of Health andHuman Services under Contract N01-CO-74102 with Program Re-sources, Inc.

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7058 Immunology: Muegge et al.

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