Regulation of tumor necrosis factor alpha transcription in

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1498-1506

Vol. 10, No. 4

0270-7306/90/041498-09$02.00/0 Copyright ©3 1990, American Society for Microbiology

Regulation of Tumor Necrosis Factor Alpha Transcription in Macrophages: Involvement of Four KB-Like Motifs and of Constitutive and Inducible Forms of NF-KB MARTINE A. COLLART,l* P. BAEUERLE,2 AND P. VASSALLI'

Departement de Pathologie, Centre Medical Universitaire, I Ruie Michel Servet, 1211 Geneva 4, Switzerland,' and Genzentrum der Ludwig-Maximilians-Universitaet Muenchen, D-8033 Martinsried, Federal Republic of Germany2 Received 21 September 1989/Accepted 12 December 1989

This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-a) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-aL gene transcription in peritoneal exudate macrophages was accompanied by induction of DNAbinding proteins that recognized with different affinities four elements related to the KB consensus motif and a Y-box motif. We suggest that the basal level of TNF-a expression in macrophages is due to the binding of a constitutive form of NF-KB, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the KB motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-KB, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-c transcription, diminished the basal level of nuclear NF-KB. Stimulation of TNF-a transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-KB. This induced factor represents a different form of NF-KB, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-KB contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-KB to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-KB was indeed detectable in the cytosol after deoxycholate treatment. UV cross-linking experiments and gel retardation assays indicated that the inducible form of NF-KB is in a higher-order complex with other proteins.

The tumor necrosis factor/cachectin (TNF-ox) cytokine is emerging as a particularly important mediator of inflammatory responses (4). Among its pleiotropic effects, TNF-a has been shown to play a major endocrine role in the pathogen-

cyclic AMP (35), cytokines (27, 35), lectins (7), doublestranded RNA (38), LPS (1, 32), and p40-tax, the transactivator protein of human T-cell lymphotropic virus type 1 (21, 29). Protein kinase A or C is able to mediate this activation

esis of gram-negative endotoxic shock and also to modulate the metabolic activities of diverse tissues (4). Moreover, it has profound autocrine effects on macrophages, its main cellular origin, activating them and enhancing their cytotoxic potential (34). We have previously described the induction of TNF-(x transcription in mouse thioglycolate-elicited peritoneal (TG) macrophages by diverse agents such as lipopolysaccharide (LPS), gamma interferon (IFN--y), and cycloheximide (CH) (9, 10). In a recent study (32a), we have shown that two or more copies of a KB-like motif from the TNF-(x promoter region are sufficient to confer inducibility on a heterologous promoter in these cells. NF-KB is a transcription factor that mediates signal transduction between cytoplasm and nucleus in many cell types, and a number of genes contain NFKB-binding sites that might control their inducible expression (1, 18). These binding sites have been shown to be necessary for transcription of the immunoglobulin kappa light-chain gene (20, 28), human immunodeficiency virus (14, 17, 25). IFN-3 (18), and the interleukin-2 receptor a-chain (21, 23, 29) genes. In uninduced situations, NF-KB is sequestered in the cytoplasm, and its activation involves a posttranslational process in which NF-KB apparently dissociates from an inhibitory protein called IKB and moves to the nucleus (1, 2). The activation of NF-KB can be induced by a variety of different agents such as phorbol esters (1, 32),

(36).

*

Here we show that in thioglycolate exudate peritoneal (TEP) macrophages, LPS, CH, and IFN-y induce the activation of NF-KB, which can bind with different affinities to four KB-like elements in the TNF-ot promoter. Our results add to the increasing number of studies that implicate NF-KB as a mediator of signal transduction leading to induction of gene transcription. Moreover, we present evidence supporting the notion that the basal level of transcription of some inducible genes, such as that of TNF-cx in macrophages, is associated to the presence of a previously unrecognized constitutive form of NF-KB. MATERIALS AND METHODS Cells. Total TEP cells, which consist to a large extent of activated macrophages (22) and will therefore be referred to as TEP macrophages, were prepared from 3-month-old CBA/CA mice 4 days after a single intraperitoneal (i.p.) injection of 1.5 ml of aged thioglycolate broth (Difco Laboratories). Stimulated TEP macrophages were obtained by injecting the agents in the peritoneal cavity of the mice at given times before collecting the TEP macrophages. Cells were collected and washed once in phosphate-buffered saline before preparation of extracts. The 70Z/3 pre-B-cell line (kind gift of F. Melchers, Basel Institute for Immunology) was cultivated in RPMI 1640 supplemented with 10% fetal calf serum and 50 ,uM ,B-mercaptoethanol and was stimulated for 90 min with CH (10 p.g/ml) and LPS (1 p.g/ml).

Corresponding author. 1498

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Nuclear run-on analysis. Nuclei were isolated at the indicated times as described (10). After in vitro elongation, the same amount of labeled RNA from each reaction was hybridized to purified DNA fragments from the TNF locus immobilized on nitrocellulose (10). Preparation of nuclear extracts. Nuclei were prepared by breaking cells swollen in a hypotonic buffer [10 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.9), 0.75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.1 mM ethylene glycol-bis(P-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 10 mM KCI, 1 mM dithiothreitol (DTT), 5 mg of powdered nonfat milk per ml, 0.5 mM phenylmethylsulfonyl fluoride-1% (wt/vol) aprotinin, 50 ,ug each of pepstatin A, leupeptin, chymostatin, and antipain per ml] with three strokes of a B pestle in a Dounce homogenizer. Some preparations also contained 78 ,ug of benzamidine per ml to further prevent proteolytic degradation. The nuclei were then lysed as described by Gorski et al. (13) in the presence of 5 ,ug each of pepstatin A, leupeptin, chymostatin, and antipain per ml. Nuclear extracts were precipitated for 20 to 60 min on ice by addition of 0.3 g of ammonium sulfate per ml and then suspended in and dialyzed for 2 to 4 h against 25 mM HEPES (pH 7.9)-0.1 mM EDTA-40 mM KCl-10% glycerol-1 mM DTT. Cytoplasmic extracts were prepared from the supernatant obtained after the nuclei were pelleted as described previously (1). Protein concentration was assessed by the Bio-Rad Bradford assay. Mobility shift electrophoretic assay. Fragments from the TNF-(x locus (30) were subcloned into pUC19 and transformed into the bacterial DH1 strain. Fragments were labeled with the Klenow fragment of DNA polymerase I after restriction enzyme digestion. For binding reactions, 0.1 to 10 ng of end-labeled fragment was mixed with 12 jig of nuclear extract protein in a total volume of 20 ,ul containing 25 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 12% (vol/vol) glycerol, and 40 mM KCl. A 2-,ug amount of poly(dI-dC) (Pharmacia) and 0.5 ,ug of sheared singlestranded Escherichia coli DNA were added to all reactions. Binding was performed for 30 min at 20 to 25°C. The reaction mixtures were then loaded on 4% polyacrylamide gels in low-ionic-strength buffer (0.25 x TBE) and run for 3 h in the cold with recycling. In the case of binding with cytoplasmic extracts, the reactions were made to 0.6% sodium deoxycholate after 30 min, followed by the addition of 1.2% Nonidet P-40 (1). G-methylation interference. The DNA probe was labeled on either strand and partially methylated with dimethyl sulfate as described previously (24). These labeled and methylated probes were used in a typical 160-,u binding reaction with a 100-fold increase of probe. Free and complexed DNAs were separated on a nondenaturing gel, electrotransferred onto DE81 chromatography paper (Whatman, Inc.), eluted, and cleaved with piperidine (24). Equal amounts of radioactive materials were resolved in sequencing gels. UV cross-linking of nuclear proteins. The TNF -510 KB template (5'-CTCAAACAGGGGGCTTTCCCTCCTCAAT ATCATGTC-3') was annealed to a primer (5'-GACATGAT ATTG-3') and filled in by the Klenow fragment of DNA polymerase I with bromodeoxyuridine triphosphate and 32Plabeled dCTP. Then 106 cpm of probe and 120 ,ug of nuclear extract were cross-linked with UV light (305 nm) for 30 min after the binding reaction was completed, digested for 30 min at 37°C with 1 U of micrococcal nuclease (Pharmacia) and 2 ,ug of DNase (RQI; Promega Biotec), and analyzed by

NF-KB AND TNF-a TRANSCRIPTION

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels. RESULTS In vivo induction of TNF-a transcription. We have previously described the induction of TNF-ot transcription by LPS, CH, and IFN-y in TG macrophages that had been allowed to adhere in vitro for 12 to 18 h (9, 10). TG macrophages were also stimulated to transcribe the TNF-ot gene when these inducing agents were injected directly in the peritoneal cavity of mice bearing a peritoneal exudate (Fig. 1A). Nuclear run-on analysis along the TNF locus showed a low basal transcriptional level in the resting TEP cells, which consist mostly of macrophages (22) (Fig. 1A, lane C), and a strongly increased transcription in TEP macrophages stimulated by an i.p. injection of either CH, LPS, or IFN-y for times chosen according to the observations previously made in vitro (9, 10). This experiment also showed that the density of polymerases was uniform along the TNF-a gene before or after induction of transcription by CH, LPS, or IFN-y, since the same amount of labeled RNA hybridized to fragments of similar lengths distributed along the TNF-ot gene. Moreover, no transcription was detected upstream of the TNF-ot gene or in the adjacent lymphotoxin gene (2738 to 3301). Inducible nuclear proteins bind to KB and Y-box DNA motifs present upstream of the TNF-a gene. Gel retardation assays were systematically performed with the various DNA fragments A to M (Fig. 1B), covering the whole region situated between the 3' end of the lymphotoxin gene and the TNF-a cap site, using nuclear extracts from resting TEP macrophages or from CH-. LPS-, and IFN-y-stimulated TEP macrophages (not shown). This analysis revealed a number of binding activities to all of the DNA fragments. Inducible binding activities, however, were observed only with fragments -695 to -432 (fragment K in Fig. 1B) and -316 to -161 (fragment M in Fig. 1B) (Fig. 2), and weaker activities also were observed with fragment -941 to -849 (fragment B in Fig. IB) (not shown). They were also observed with the overlapping fragments D, E, and H. The two first fragments contain cis-acting upstream DNA regions (-655 to -451 and -301 to -241) necessary for the LPS-mediated increased transcription of the TNF-Qx gene; these regions were identified by transfection experiments in macrophages, and LPSinduced nuclear proteins were shown to bind to DNA fragments spanning these regions (32a). The three inducing agents gave similar patterns of gel retardation (Fig. 2). Fragment -695 to -432 (Fig. 2A) showed two strongly retarded complexes, called Ia and lb, which were detected only with extracts from stimulated cells. The two less retarded complexes II and IV correspond to DNA-binding proteins also present in resting TEP macrophages (the protein-DNA complex migrating slightly faster than complex II is probably a proteolytic product of either complex Ia or lb, since it was not seen with nuclear extracts prepared in the presence of the protease inhibitor benzamidine [not shown]). G-methylation interference experiments (Fig. 3A) showed that the factors present in complexes Ia, lb, and II made similar contacts to a single region of the DNA fragment, which corresponds to a KB consensus sequence (-513 to -503) (see also Fig. 4B, on which this sequence on a shorter DNA fragment shows a better resolution). This result is in accord with our previous observation that an oligonucleotide containing the KB motif could compete for complexes Ia, lb, and II (32a). This pattern of

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-4yi -ICII -321 I c63 FIG. 1. Nuclear run-on analysis of resting and stimulated TEP macrophages (A) and map of the TNF locus (B). (A) Nuclei were isolated from resting TEP macrophages (lane C) or from TEP isolated 45 min after an i.p. injection of 500 ,ul of CH (200 ,ug/ml) (lane CH), 30 min after an i.p. injection of 500 p.1 of LPS (20 ,ug/ml) (lane LPS), or 60 min after an i.p. injection of 500 ,u1 of IFN-y (5 U/ml) (lane IFN). Elongation was allowed to proceed in the presence of 32P-labeled UTP; the amounts of 32P-labeled RNA synthesized by the nuclear preparations were equivalent, and identical amounts were hybridized to the indicated fragments of the TNF locus. The 322-base-pair Pi'lII fragment of pUC19 was used to appraise background hybridization. (B) At the top is a map of the murine TNF locus. Positions of exon-intron boundaries (according to Semon et al. [30] and corrected according to EMBL/GenBank data library accession no. Y00467) are indicated. Below, the fragments used in binding reactions (see text) are indicated by roman capitals, and their positions with respect to the cap site of TNF-a (32a) are indicated.

protein-DNA contacts was identical with that of nuclear extracts obtained from TEP macrophages exposed to each of the three inducing agents. The pattern of G-methylation interference observed with complex IV showed proteinDNA contacts at a nucleotide sequence (-659 to -649) that corresponds to a consensus found in the upstream region of several lymphokines and cytokines (33) and for this reason is called cytokine 1 (CK-1). Formation of this complex was prevented by an oligonucleotide containing the CK-1 but not the KB motif (32a). Fragment -316 to -161 formed a single complex with nuclear extracts of stimulated as well as of resting TEP macrophages (Fig. 2B), but the amount of complex formed with extracts of stimulated TEP macrophages was about three times greater (probably even more for IFN-y stimulation). G-methylation interference localized the protein-DNA contacts between nucleotides -259 to -247 (Fig. 3B), which corresponds to a consensus sequence known as Y-box motif (11). The binding properties of

nuclear extracts to a DNA sequence contained within fragment -941 to -849 are discussed below. The nuclear factors in macrophages that bind to the TNF-ot KB motif correspond to at least two forms of NF-KB. The following lines of evidence indicate that the TEP macrophage KB-binding factors, even though they form complexes of different electrophoretic mobilities on native gels with a DNA fragment containing the TNF-ax KB motif, cannot be distinguished from the protein complex called NF-KB as it is found in pre-B and B lymphocytes. (i) Binding to a DNA fragment containing the immunoglobulin K light-chain gene KB motif of nuclear extracts from LPS- and CH-induced 70Z/3 cells, a pre-B-cell line that is the original source of NF-KB (32), was completely prevented in the presence of an unlabeled oligonucleotide containing the TNF-Ot KB motif, as shown by a gel retardation assay (Fig. 4A, lanes 1 and 2), but was unaffected by the presence of similar amounts of an irrelevant unlabeled oligonucleotide (not shown). (ii) Nu-

NF-KB AND TNF-a TRANSCRIPTION

VOL. 10, 1990

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IFN-y. Nuclear extracts were prepared from restin ig TEP macrophages (lane C) or TEP macrophages stimulated as di escribed in the legend to Fig. 1. Each extract (12 ,ug of protein) was incubated with an end-labeled Sacl (-695)-to-NcoI (-432) (A) or to-FniuDII (-161) fragment (B). The binding reactic rated on native 4% polyacrylamide gels. Specific c sessed by competition with excess cold fragment) zare marked by arrows. A complex in panel A is not indicated beca use it was not observed with nuclear extracts prepared from cell Is lysed in the presence of benzamidine (see Materials and Method s).

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clear extracts from induced 70Z/3 cells and stil mulated TEP macrophages incubated with a labeled oligonuicleotide containing the TNF-a -510 KB motif formed two Iprotein-DNA complexes (called I and TT) of similar electroph oretic mobility (Fig. 4A, lanes 3 and 4). (iii) By G-methyla tion interference, these two protein-DNA complexes I and IT formed by nuclear extracts of either stimulated 70Z/3 or sti mulated TEP macrophages showed similar contacts to the TNF-a -510 KB motif (Fig. 4B). (iv) In the presence c)f millimolar concentrations of GTP, the nuclear extracts froi m resting and stimulated TEP macrophages had increased afffinity for the TNF-a -510 KB motif, as can be seen in Fig. 4C" for complex TI (lanes 1 to 4). Complexes Ta and Tb (re sulting from resolution of complex I by analysis on native 6 % polyacrylamide gels) were also GTP sensitive but at low(er concentrations of GTP (not shown). This effect of GTP c oncentration on DNA binding is a known property of NF -KB (19). (v) Deoxycholate treatment of cytoplasmic extracts from resting TEP macrophages revealed binding activities that formed two complexes with electrophoretic mobilities identical to those obtained with nuclear extracts from induc ed cells only, i.e., complexes Ia and Tb, but did not form connplex II (Fig. 4C, lanes 5 and 6). These latent binding activitie s were not or barely detectable in cytoplasmic extracts fro m stimulated TEP macrophages (not shown). The presenc e of a latent binding activity in cytoplasmic extracts from uniinduced cells is a property of NF-KB (1); this activity appal rently results from the release by the detergent of NF-KB fron n its inhibitor IKB, which maintains the factor in an inactiv e form and a cytoplasmic localization (1). Thus, by all of these criteria, the factors prn esent in TEP macrophage nuclear extracts and able to bind tl he TN F-Ot KB motif at -510 are indistinguishable from NF-K cB present in stimulated pre-B- and B-lymphocyte nuclear extracts. We will show below that purified NF-KB indeed bin ids this motif. However, a puzzling observation is that l protein-DNA complexes of different electrophoretic mobilitil es on nondenaturing gels (called Ia, Tb, and II) were forr ned with the TNF-cx KB -510 oligonucleotide and nucleai extracts of

likely that complexes Ta and lb resulted from partial proteolysis of complex Ia (see below). For differences between complexes I and TI, a possible explanation is that proteins other than NF-KB are additionally contained in the slowermigrating complex I. Thus, to determine whether in all of these complexes only NF-KB was bound to the DNA or whether other DNA-bound proteins could be detected, we analyzed on denaturing sodium dodecyl sulfate-polyacrylamide gels the proteins bound to the KB motif that could be covalently linked to the DNA by UV cross-linking of the protein-DNA complexes. This was performed with complexes obtained from nuclear extracts of resting and stimulated TEP macrophages and from deoxycholate-treated cytoplasmic extracts of resting TEP macrophages. All of the protein-DNA complexes (see Fig. 5A for the analysis on native gels) contained a DNA-bound protein of approximately 50 kilodaltons (kDa) (Fig. SB). This value corresponds to the molecular weight recently reported for the DNA-binding subunits of purified NF-KB (17). An additional DNA-bound protein of about 100 kDa was found in the more slowly migrating complexes Ta and lb; its significance in relation to the known subunit composition of purified NF-KB and the possible interpretation of the difference in migration of protein-DNA complexes apparently containing the same DNA-bound protein will be discussed below. Since dexamethasone has been shown to strongly decrease TNF-a transcription in macrophages (5, 10), nuclear extracts of dexamethasone-treated TEP macrophages were also tested for DNA-binding activity on the TNF-a -510 oligonucleotide. The binding activity of the extracts was reduced (Fig. SA), and the amount of 50-kDa protein bound was equally reduced (Fig. SB). The TNF-a promoter contains four variants of the KB motif that bind NF-KB with different affinities. On the basis of sequence similarities, we have previously proposed that the CK-1 motif at position -655 and a similar motif at position -210 could be somewhat related to the KB motif and that the sequence from -862 to -852 in the TNF-(x promoter region could represent a closely related variant of the KB motif (32a). In support of this idea are two observations: (i) as mentioned earlier, nuclear extracts of stimulated TEP macrophages contained inducible binding activities to a fragment containing this latter element (-941 to -849) (not shown); and (ii) an oligonucleotide containing the CK-1 motif competed with low affinity for binding of NF-KB on the KB motif of the -695 to -432 fragment (32a). To test the possibility of a relationship between the -513 to -503 KB motif and these other three DNA sequences, we performed gel retardation assays with nuclear extracts of stimulated or resting TEP macrophages and four synthetic oligonucleotides, each containing one of the above-mentioned sequences (Fig. 6A). All four oligonucleotides interacted with various binding activities present in nuclear extracts of stimulated TEP macrophages, one or two (depending on the oligonucleotide) of which were absent in nuclear extracts of resting TEP macrophages (arrows in Fig. 6A). Moreover, all of the binding on each oligonucleotide could be competed for by the other three oligonucleotides except for the very abundant complexes formed with extracts from resting and stimulated TEP macrophages and the -850 and -655 oligonucleotides (dots in Fig. 6A), which thus appear to have no relation to NF-KB. In all cases, the strongest competitor for binding on the four oligonucleotides

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the -510 motif, followed with decreasing affinity by the -850, -656, and -210 motifs (see Fig. 6B for competition of binding with the -510 oligonucleotide; other data not shown). These results suggest that all of these DNA sequences can be bound by NF-KB, albeit with different affinities. To confirm this, we performed a binding analysis with purified NF-KB and the four oligonucleotides containing the putative TNF-ct KB motifs, as well as a fragment containing the

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NF-KB AND TNF-a TRANSCRIPTION

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FIG. 4. Identification of NF-KB in TEP macrophage extracts. (A) Plasmid J10 (28) was digested by HindIlI and Sall, releasing a 46-mer containing the immunoglobulin K light-chain KB motif and polylinker sequences from pUC13. A 10-ng sample of labeled immunoglobulin KB fragment was incubated with 12 p.g of extracts from CH- and LPSinduced 70Z/3 cells in the presence of 3 mM GTP in the absence (lane 1) or presence (lane 2) of a 50-fold molar excess of the TNF -510 KB oligonucleotide (see legend to Fig. 6). This latter oligonucleotide was labeled and incubated with 12 ,ug of nuclear extracts from CHstimulated TEP macrophages (lane 3) or 70Z/3 cells (lane 4). (B) Complexes I and II from lanes 3 and 4 of panel A were analyzed by G-methylation interference. (C) Labeled -510 TNF-a oligonucleotide was incubated with 12 ,ug of nuclear extracts from resting TEP macrophages in the absence (lane 1) or presence (lane 2) of 3 mM GTP, 12 ,ug of nuclear extracts from CH-stimulated TEP macrophages in the absence (lane 3) or presence (lane 4) of 3 mM GTP, or 24 pLg of cytoplasmic extract from resting TEP macrophages without (lane 5) or with addition of (lane 6) deoxycholate to a final concentration of 0.6%; Nonidet P-40 was added at the end of the binding reaction to 1.2% (lane 6). The reactions were analyzed on a native 6% polyacrylamide gel. In lanes 3 and 4, the observed protein-DNA complex migrating slightly faster than complex II is probably a proteolytic product of either complex Ia or lb, since it was not seen with nuclear extracts obtained in the presence of the protease inhibitor benzamidine (see above).

FIG. 5. UV cross-linking of TEP macrophage nuclear extracts to the TNF -510 KB oligonucleotide. Nuclear extracts from TEP macrophages treated i.p. for 90 min with 500 ,ul of dexamethasone (2 x 10-9 M) (lane 1), resting TEP macrophages (lane 2), TEP macrophages stimulated for 45 min with 500 ,u1 of CH (200 ,ug/ml) (lane 3), or deoxycholate-treated cytoplasmic extract from resting TEP macrophages (lane 4) were incubated with a labeled TNF -510 KB oligonucleotide. The binding reaction mixtures were then crosslinked for 30 min with UV light. A sample of these mixtures was analyzed on a native 6% polyacrylamide gel (A), and the remaining mixtures were digested with DNase and micrococcal nuclease and then run on a 10% sodium dodecyl sulfate-polyacrylamide gel (B).

analyzed on native 6% polyacrylamide gels (not shown). Complex lb probably resulted from progressive partial proteolysis of complex Ia, as suggested by the respective amounts of the two complexes after various times of incubation. DISCUSSION A systematic binding analysis of the TNF-a promoter region with nuclear extracts from inactivated, resting, or stimulated TEP macrophages has shown that modulation of TNF-a transcription parallels the modulation of binding activities on four elements related to the KB consensus motif and on a Y-box motif. The KB-binding activities are indistinguishable from NF-KB-binding activities found in pre-B and B cells by the following criteria: (i) contact to DNA was identical, (ii) DNA-binding affinity was increased in the presence of GTP, (iii) a DNA-binding subunit of 50 to 55 kDa was observed, and (iv) a similar DNA-binding factor was found in deoxycholate-treated cytoplasmic extracts of resting cells.

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FIG. 6. Demonstration that four variants of the KB motif are contained in the TNF-a promoter. Four double-stranded oligonucleotides were used (the putative NF-KB-binding motifs are underlined): oligonucleotide -850, 5'-GATCTGGGAGGGGAATCCTTG GAA-3'; oligonucleotide -655, 5'-TTGTGAGGTCCGTGAATTC CCAGGGCTGAGTTCA-3'; oligonucleotide -510, 5'-AGCTCAA ACAGGGGGCTTTCCCTCCTC-3'; and oligonucleotide -210, 5'GATCCGGAGGAGATTCCTTGATGG-3'. (A) End-labeled oligonucleotides were incubated with 12 ,ug of extracts from resting (-) or CH-stimulated (+) TEP macrophages. Because the binding reactions with the four oligonucleotides were run on separate gels, the mobilities of the protein-DNA complexes formed with the different oligonucleotides cannot be directly compared. (B) The -510 labeled oligonucleotide was incubated with 12 ,ug of CHstimulated extracts, and a 500-fold excess of cold oligonucleotides was added: Y oligonucleotide (irrelevant) (lane 1), -850 (lane 2), -655 (lane 3), -510 (lane 4), and -210 (lane 5). The Y oligonucleotide (-250) is 5'-AAAAACCTCCTGATTGGCCCAGATTGCCAC AGATCCTGGGTGGGGG-3'.

NF-KB is present in the nuclei of resting TEP macrophages (which have a low basal transcriptional level of the TNF-a gene) at a low level (for this reason, we call it constitutive NF-KB) and as latent inducible forms in their cytoplasm (inducible NF-KB). These distinct NF-KB forms lead to the formation of two main protein-DNA complexes of different electrophoretic mobility on native gels: the fastmigrating complex TI (containing constitutive NF-KB) and the slower-migrating complex Ia (and its proteolytic variant Ib) containing inducible NF-KB. In both slow- and fastmigrating complexes, a 50-kDa protein was bound to the DNA and could be cross-linked by UV. In the slowermigrating complexes, an additional 100-kDa protein was detected. It may represent a homodimer of the 50-kDa protein. Indeed, NF-KB purifies as a heterotetramer containing a DNA-binding 50-kDa homodimer and an additional non-DNA-binding 65-kDa homodimer. Moreover, in vitro experiments showed that NF-KB can be converted by IKB into a cytoplasmic latent form only if the 65-kDa protein is present (2a). Thus, in all probability, the slower-migrating

inducible complexes (Ta and Tb) correspond to intact or partially proteolyzed forms of a heterotetramer containing

FIG. 7. Binding analysis with purified NF-KB. Purified human NF-KB (0.5 ,ul) was incubated with a labeled immunoglobulin KB fragment (see Fig. 6) (lane 1) and the following labeled oligonucleotides: -510 TNF-a KB (lane 2), -850 TNF-a KB (lane 3), -655 TNF-a KB (lane 4), and -210 TNF-a KB (lane 5). Binding reactions were performed in the presence of 1% Nonidet P-40 and 20 ,ug of bovine serum albumin in 10 mM Tris (pH 7.5)-75 mM NaCl-6% glycerol-1 mM EDTA-1 mM DTT-0.1 mM phenylmethylsulfonyl fluoride. The reactions were loaded on a 4% native polyacrylamide gel, which was run at room temperature in a Tris acetate buffer.

the 65-kDa protein. Indeed, both of these complexes (but not complex II) are formed with deoxycholate-treated cytoplasmic extracts from resting TEP macrophages and with purified heterotetrameric NF-KB. Previous studies mainly assigned to NF-KB one inducible binding activity that forms a single complex on nondenaturing gels with KB motifs (1, 19, 32). The two different inducible forms of NF-KB that we observe (complexes Ta and Tb) could be resolved only in 6% nondenaturing gels, not in 4% gels (compare Fig. 4A and C). It is likely that in previous experiments either they were not resolved or there was less proteolytic degradation. In one previous report, KB-binding activities in nuclear extracts from inflammatory peritoneal macrophages were analyzed by gel retardation, and two complexes of different mobility were also observed (14). The new constitutive form of NF-KB that we describe could correspond to the 50-kDa DNA-binding homodimer only. Absence in this form of the additional 65-kDa protein would explain why this activity was constitutively present in the nucleus and not stored in the cytoplasm in resting TEP macrophages complexed with lKB (2a). This form of NF-KB binds to the TNF-ot -510 KB motif with stronger affinity than to the KB motif of the immunoglobulin K enhancer (compare lanes 1 and 4 of Fig. 4A), which might explain why it was not seen previously with K-enhancer-derived DNA probes (1, 19, 32). An observation that remains puzzling if the cross-linked 100-kDa protein is indeed the homodimeric 50-kDa DNAbinding protein of NF-KB is why both subunits of the homodimer can be cross-linked only in the more retarded complexes Ta and Tb, since it should also be present in complex TI. A possible explanation is that the presence of the 65-kDa homodimer favors cross-linking of both 50-kDa monomers to the DNA or to each other. Binding of NF-KB has been demonstrated to be important in the activation of K light-chain gene transcription during B-lymphocyte development (31). The importance of NF-KB binding in the activation of TNF-ot transcription in macro-

VOL. 10, 1990

phages is highlighted by previous functional studies which showed that two or more copies of the TNF-ot -510 KB motif are sufficient to confer inducibility on a heterologous promoter and that three or more copies are required for optimal inducibility (32a). In this study, we show that four elements in the TNF-a promoter are potential binding sites for NFKB, with very different affinities: the site at -510 is bound as strongly as the immunoglobulin KB sequence, and the site at -850 is also well bound, whereas the sites at -655 and -210 are only weakly recognized. However, though multiple copies of a KB motif seem to be required for activation of the TNF-a gene in macrophages, the relative importance of these four different KB sites in the TNF-a promoter is not yet clear. Indeed, the four sites are not just four variants of KB motifs with more or less affinity to NF-KB: the -655 and -850 sites can also be bound with high affinity by some other protein(s) constitutively present in macrophage nuclei and not related to NF-KB, since competition with oligonucleotides containing other NF-KB-binding sites will not prevent their binding to these two motifs. Thus, different factors could be bound to the four KB motifs in vivo, and this is actually what might be required for efficient expression in macrophages: cooperation between different factors bound to a variety of KB-like sequences or even to other sites such as Y-box motifs, rather than cooperation between many bound NF-KB molecules. In accord with this hypothesis is our previous observation that levels of inducibility brought about by two copies of the highest-affinity NF-KB site (-510 site) on a heterologous promoter and a nested deletion of the TNF-a promoter containing only one copy of the weakest affinity NF-KB site (-210 site) and a Y-box site are comparable (32a). We show in this study that the binding activity on the Y box is one of the authentic CCAAT-box-binding proteins, some of which have been shown to activate transcription (11). The addition of a Y box to a KB motif in a basal promoter could then synergize, maybe tissue specifically, with the NF-KB-dependent transcriptional activity. Previous studies suggesting that the Y box has a particular importance for the macrophage-specific expression of major histocompatibility complex class II genes argue in favor of such a model (37). NF-KB seems to be a major intracellular transducer of a variety of external signals in many, if not all, cell types (18). However, the requirement for cooperative interaction between NF-KB-binding sites and other motifs could be different in different cell types and may be an important element ensuring that all NF-KB-regulated genes are not expressed simultaneously in all cell types in vivo. For instance, genes such as lymphotoxin and interleukin-2 expressed in lymphocytes contain a single copy of the KB motif and are not expressed in mouse macrophages, maybe because of the lack of additional elements such as a Y box or other KB/CK-1 motifs. Among the increasing number of genes for the transcriptional induction of which NF-KB appears to be involved are genes playing a role in inflammatory and immune responses, such as major histocompatibility complex class I and 11 (3, 6, 16). IFN-3 (20, 38), and cytokine or lymphokine (15) genes. Thus, our observation that glucocorticoids repress NF-KB activity in macrophages, if it extends to other cell types, may be relevant to understanding their role as immunosuppressors. We have detected basal transcription of the TNF-a gene in a number of cell types (macrophages, 70Z/3 pre-B cells, and L929 fibroblasts), all of which contain the constitutive form of NF-KB in their nuclei (not shown). This form, which we

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postulate to contain only the DNA-binding 50-kDa homodimer and not the 65-kDa subunits necessary to bind NF-KB to its cytoplasmic inhibitor (2a), as discussed above, could therefore be responsible for the basal transcription of the TNF-a gene. The observation that dexamethasone, which decreases TNF-a transcription, also decreases this constitutive form in macrophages argues in favor of this hypothesis. Strong induction of TNF-a transcription would then result from an increase of the NF-KB-binding activity, which would derive from the cytoplasmic inducible form, which contains the 65-kDa subunits. In this context, it should be noted that other agents which induce TNF-a transcription, and which have not been analyzed here, such as cyclic AMP (10), phytohemagglutinin (unpublished observations), TNF-a itself (8), mitogens (26), and viral infection (12), have all been shown to lead to NF-KB activation (1, 7, 27, 32, 35). It will be interesting to find out whether other genes constitutively expressed at low levels and inducible by NF-KB also contain KB motifs that bind with high affinity to the constitutive form of NF-KB. ACKNOWLEDGMENTS We thank S. Nedospasov for a genomic clone of the mouse TNF locus and M. Lenardo for the J-10 plasmid. We thank C. Magnin and C. Briottet for excellent technical assistance and U. Schibler, W. Reith and S. Satola for helpful discussions. We are grateful to D. Belin for his careful examination of the manuscript. This work was supported by grant 3.650.87 from the Swiss National Science Foundation. LITERATURE CITED 1. Baeuerle, P. A., and D. Baltimore. 1988. Activation of DNAbinding activity in an apparently cytoplasmic precursor of the NF-KB transcription factor. Cell 53:211-217. 2. Baeuerle, P. A., and D. Baltimore. 1988. IKB: a specific inhibitor of the NF-KB transcription factor. Science 242:540-546. 2a.Baeuerle, P. A., and D. Baltimore. 1989. A 65-kD subunit of active NF-KB is required for inhibition of NF-KB by 1 KB. Genes Dev. 3:1689-1698. 3. Baldwin, A. S., and P. A. Sharp. 1988. Two transcription factors, NF-KB and H2TF1, interact with a single regulatory sequence in class I major histocompatibility complex promoter. Proc. Natl. Acad. Sci. USA 85:723-727. 4. Beutler, B., and A. Cerami. 1988. The history, properties and

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