©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the TSG-6 Gene by NF-IL6 Requires Two Adjacent NF-IL6 Binding Sites (*)

(Received for publication, September 1, 1994; and in revised form, December 7, 1994)

Lidija Klampfer (§) Selina Chen-Kiang (1) Jan Vilcek (¶)

From the Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016 and the Brookdale Center for Molecular Biology, Mt. Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) encodes a protein expressed during inflammation. We have previously shown that transcription factors of the NF-IL6 and AP-1 families cooperatively modulate activation of the TSG-6 gene by TNF or interleukin 1 (IL-1) through a promoter region that contains an NF-IL6 site (-106 to -114) and an AP-1 element (-126 to -119). In this study we report the identification of an additional NF-IL6 site (NF-IL6*) located at positions -92 to -83. Footprinting and electrophoretic mobility shift assay suggested that NF-IL6 binds with higher affinity to the newly identified NF-IL6* site than to the earlier identified promoter-distal NF-IL6 site and that the two sites cooperate in binding NF-IL6. TNF and IL-1 stimulate specific binding of nuclear proteins to the NF-IL6* site more efficiently than to the promoter-distal NF-IL6 site. Moreover, a mutation in the NF-IL6* site abolished transactivation of the TSG-6 promoter by NF-IL6 despite the presence of the intact promoter-distal NF-IL6 site. A mutation in the promoter-distal NF-IL6 site also greatly decreased activation of the TSG-6 promoter by NF-IL6. We conclude that the two NF-IL6 sites are functionally interdependent in the activation of the TSG-6 gene.


INTRODUCTION

Tumor necrosis factor (TNF)^1-stimulated gene 6 (TSG-6) encodes an inducible secreted protein that shows partial structural homology to members of a family of hyaluronan-binding proteins(1) . A role for TSG-6 in inflammation is suggested by the presence of high concentrations of TSG-6 protein in the synovial fluid of arthritis patients(2) . Expression of TSG-6 is tightly regulated at the level of transcription, with its synthesis rapidly induced by the proinflammatory cytokines TNF and IL-1 and by bacterial lipopolysaccharide in normal human fibroblasts, mononuclear cells, chondrocytes, and synovial cells(2) . TSG-6 is a primary response gene because its transcriptional activation by TNF in human FS-4 fibroblasts was not reduced in the presence of cycloheximide(3) . Earlier studies have shown that the regulation of the native TSG-6 promoter by cytokine-generated signals is complex. The region between -163 and -58 confers inducibility to the TSG-6 gene by TNF and IL-1(3) , and an NF-IL6 site (-106 to -114) within this region is indispensable for activation by both cytokines(4) . The NF-IL6 site mediates activation of the TSG-6 promoter by NF-IL6(4) , the major member of the human CCAAT/enhancer-binding protein family(5) . As predicted from earlier studies with synthetic promoters(6, 7) , activation of the native TSG-6 promoter by NF-IL6 is modulated by the ratio of the activator to inhibitor isoforms of NF-IL6(4) . An AP-1 site within this cytokine-responsive region (-126 to -119), adjacent to the NF-IL6 site, is also essential for activation by TNF, and it enhances induction by IL-1(4) . Activation via the AP-1 site is mediated by Fos/Jun family members. However, the AP-1 site also binds the inhibitor isoform of NF-IL6, which can inhibit AP-1 site-mediated transcription of the TSG-6 gene(4) .

NF-IL6 (also called IL-6DBP, AGP/EBP, C/EBPbeta, rNF-IL6, or CRP-2) mediates responses to IL-6, TNF, and IL-1, important in inflammation, in the acute phase response(8, 9, 10) , and in viral infections(11) . Analogous to its rodent counterpart, the human NF-IL6 gene lacks introns, but it encodes three proteins (NF-IL6-1, NF-IL6-2, and NF-IL6-3) that are translated from in-frame AUGs of the same mRNA species(6, 7) . NF-IL6 dimers regulate target genes by binding through the basic leucine zipper regions to specific NF-IL6 recognition sequences(9, 12) . NF-IL6 can function as an activator or inhibitor, depending on the ratio of activator to inhibitor isoforms(6) . Stimulation by cytokines and retinoic acid leads to an increase in the activator forms, suggesting that alteration in the ratio of activator to inhibitor forms of NF-IL6 is one of the means by which this transcription factor mediates responses to cytokines(13) . In addition, NF-IL6 associates in vitro with proteins of other transcription factor families, such as NF-kappaB(14, 15, 16) , the glucocorticoid receptor(17) , C/ATF(18) , and AP-1(7) , raising the possibility that the biological functions of NF-IL6 are modulated by cross-family protein-protein interactions. The possibility that interactions between NF-IL6 and AP-1 play a physiologic role is supported by the finding that the interaction between NF-IL6 and Jun in vivo is regulated by IL-6. (^2)

Recent evidence from studies of promoters from several IL-1- and IL-6-responsive genes shows that multiple NF-IL6 recognition sites are functionally important for their activation by cytokines(11, 17, 20, 21) . In this report we identify a hitherto uncharacterized NF-IL6 binding site in the TSG-6 promoter. We show that this site binds NF-IL6 more efficiently than the previously identified NF-IL6 site and that the two NF-IL6 sites are functionally interdependent in mediating the activation of the TSG-6 gene. This functional interdependence distinguishes the interaction of the two adjacent NF-IL6 sites in the TSG-6 promoter from some other promoters in which two NF-IL6 binding sites are either mutually antagonistic (11, 17) or act independent from each other(20, 21) .


EXPERIMENTAL PROCEDURES

Cell Cultures and Cytokines

Normal human FS-4 fibroblasts were grown in Eagle's minimal essential medium with 5% fetal calf serum. HeLa cells were grown in Eagle's minimal essential medium with 10% fetal calf serum. Escherichia coli-derived recombinant human TNF-alpha was provided by M. Tsujimoto of the Suntory Institute for Biochemical Research, Osaka, Japan. E. coli-derived recombinant human IL-1alpha was a gift of A. Stern and P. Lomedico, Hoffmann-La Roche, Nutley, NJ.

Plasmids

The reporter plasmid pBBCAT contains the -165 to -78 region of the TSG-6 promoter, linked to the CAT gene(3) . 5`-deletions of pBBCAT (see Fig. 3A) were generated by the exonuclease III/S1 nuclease method(4, 22) . Site-directed mutagenesis of the TSG-6 promoter was performed according to Deng and Nickoloff (23) and confirmed by sequencing(24) . Mutagenic primers were designed to introduce mutations into the promoter-distal NF-IL6 site and the NF-IL6* site (Fig. 1). A selection primer (CCAGTGCCAcGCgTGCATGCCTG) was used to convert the unique HindIII restriction site in the pBBCAT vector into a MluI site(23) . The NF-IL6 expression vector (pCMV-NF-IL6) contains a full-length NF-IL6 cDNA under the control of the cytomegalovirus promoter(12) . All three NF-IL6 isoforms translated from in-frame AUGs of the same mRNA species (termed NF-IL6-1, NF-IL6-2, and NF-IL6-3) are expressed by this vector in mammalian cells(7) .



Figure 3: NF-IL6 binds to multiple sites in the TSG-6 promoter. A, schematic diagram of the -165/+74 region of the TSG-6 promoter and its 5` deletions(4) . B, GST-NF-IL6-3 fusion protein (100 ng) was incubated with end-labeled HindIII-ScaI fragments isolated from the wild-type TSG-6 promoter (pBBCAT) or from its 5`-deletion constructs schematically illustrated in A. Competition was performed with a 50-fold excess of unlabeled DNA probe. EMSA was run as described under ``Experimental Procedures.'' Arrows indicate the positions of protein-DNA complexes C1, C2, and C3, separated from the free probe (FP).




Figure 1: Mutagenic primers designed to introduce mutations (lower case letters) into the promoter-distal NF-IL6 site and the NF-IL6* site. Arrows indicate the two binding sites and their orientation.



DNase I Footprinting Analysis

Increasing amounts (20-200 ng) of GST-NFIL6-3 fusion protein(7) , which corresponds to the smallest form of the three naturally occurring NF-IL6 isoforms, were incubated with an end-labeled HindIII-ScaI fragment of the TSG-6 promoter (approximately 10,000 cpm) in a binding buffer containing 0.5 M Hepes (pH 7.9), 0.12 M EDTA (pH 8), 0.5 M KCl, and 20% glycerol. Binding reactions were carried out in a total volume of 50 µl at room temperature for 30 min, and the samples were then subjected to DNaseI digestion (0.1 units/reaction, Boehringer Mannheim) for 1 min on ice. The reactions were stopped by the addition of a buffer containing 100 mM Tris-HCl (pH 8), 100 mM NaCl, 1% sodium sarcosyl, 10 mM EDTA (pH 8), and 25 µg/ml of calf thymus DNA. After phenol-chloroform extraction, DNA was precipitated with ethanol, vacuum dried, and analyzed on a 8% urea acrylamide gel. The G+A sequencing ladder was prepared according to Maxam and Gilbert(25) .

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were prepared from FS-4 cells that were untreated or stimulated with TNF (20 ng/ml) or IL-1 (1 ng/ml) for 2 h as described previously(7) . Aliquots of 4 µg of nuclear proteins were used in each binding reaction. Recombinant GST-NF-IL6-3 fusion protein (7) was used at the concentrations indicated. Binding reactions were performed in 5 mM Tris-HCl (pH 7.5), 10 mM Hepes (pH 7.9), 50 mM NaCl, 15 mM EDTA, 5 mM dithiothreitol, and 10% glycerol at 37 °C for 15 min. Poly(dI-dC) (1 µg) was added, and the incubation was continued for 5 min before the addition of the radiolabeled DNA probe (20,000 cpm). The protein-DNA complexes formed were separated from the free probe by electrophoresis in a 6% native polyacrylamide gel in 0.25% TBE buffer (22 mM Tris base, 22 mM borate, 0.625 mM EDTA). The oligonucleotides used in EMSA, corresponding to the promoter-distal NF-IL6 site, NF-IL6* site, and AP-1 site, are shown in Fig. 2.


Figure 2: Oligonucleotides used in the electrophoretic mobility shift assay. Arrows indicate the orientation of the NF-IL6 binding sites.



Transfections and CAT Assay

HeLa cells were transfected by the calcium phosphate precipitation method(3) . Cells in 100-mm dishes at approximately 70% confluence were transfected with 20 µg of pBBCAT reporter plasmid alone or together with 1 or 5 µg of pCMV-NF-IL6. Cell lysates were prepared and assayed for CAT activity 24 h after transfection(4) . All quantifications of CAT activity were performed with the aid of the Ambis radioactive imaging system (Ambis, Inc., San Diego, CA).


RESULTS

The TSG-6 Promoter Contains Two NF-IL6 Sites

Previously we have shown that an NF-IL6 binding site (5`-TGAAGCAAA-3`, at positions -106 to -114) is essential for transactivation of the TSG-6 promoter by NF-IL6 and for its transcriptional activation by TNF and IL-1(4) . Analysis of the binding of recombinant NF-IL6 protein to the HindIII-ScaI fragment (-165 to -58) of the native TSG-6 promoter (Fig. 3A) by EMSA revealed the formation of three protein-DNA complexes, C1, C2, and C3 (Fig. 3B). The C1 complex was not formed when a 5`-deletion construct lacking the CCAAT box (PBD4) was used as a source of the probe, suggesting that C1 complex formation requires the CCAAT motif. The AP-1 site appears not to be essential for the formation of C2 and C3 complexes because the same mobility shift pattern was observed with a probe lacking the AP-1 site (PBD56). Deletion of the NF-IL6 site (pBD7) abolished the formation of the C2 complex; however, formation of the C3 complex was not affected, suggesting that there is an additional NF-IL6 binding site located downstream of the -106 to -114 NF-IL6 site. Footprinting analysis with recombinant GST-NF-IL6-3 protein localized a region protected from DNase I digestion between positions -100 and -80 (Fig. 4A). Within this region we found an NF-IL6-like sequence (5`-TTGTGTAAC-3`) spanning nucleotides -92 to -84 (Fig. 4B). The lack of protection of the previously identified promoter-distal NF-IL6 site is most likely due to its relatively lower affinity for the NF-IL6 protein (see below).


Figure 4: DNase I footprinting of the TSG-6 promoter. A, A HindIII-ScaI (-165 to -58) fragment of the TSG-6 promoter was end-labeled on the noncoding strand and incubated in the absence (lane 1) or in the presence of increasing amounts of recombinant GST-NF-IL6-3 (20, 60, 100, and 200 ng, respectively, lanes 2-5). Thereafter, DNase I was added as described under ``Experimental Procedures.'' Lane G+A shows the nucleotide sequence marker. A region protected from DNase I digestion is marked by the bracket and the nucleotide sequence within that region is shown. B, nucleotide sequence of the -140 to -61 region of the TSG-6 promoter showing positions of characterized binding sites, with the NF-IL6* site printed in boldface.



The Two Adjacent NF-IL6 Sites in the TSG-6 Promoter Bind NF-IL6 with Different Efficiencies

The newly identified NF-IL6 site at positions -92 to -84 in the TSG-6 promoter, designated NF-IL6* (5`-TTGTGTAAC-3`), differs from the NF-IL6 binding consensus sequence (T(T/G)NNGNAA(T/G)) in one position and is separated from the promoter-distal NF-IL6 site by only 13 base pairs. We prepared a synthetic oligonucleotide containing the NF-IL6* site and used it as a probe in EMSA. As shown in Fig. 5A, this site binds recombinant NF-IL6 efficiently in a concentration-dependent manner (lanes 2-4). Competition with the unlabeled promoter-distal NF-IL6 oligonucleotide (lane 5) or NF-IL6* oligonucleotide (lane 6) suggested that the NF-IL6* site competed for NF-IL6 binding more efficiently than the 5` NF-IL6 site. Supershift with antibody to NF-IL6 confirmed that the complex is NF-IL6-specific (lane 7). We compared the two NF-IL6 sites also by incubating increasing amounts of NF-IL6 protein with the same amounts of the 5` NF-IL6 or NF-IL6* oligonucleotide that were labeled to comparable specific activities (Fig. 5B). This experiment confirmed that the NF-IL6* site binds NF-IL6 more efficiently than the 5` NF-IL6 site (compare lanes 2-4 and 7-9).


Figure 5: NF-IL6 sites in the TSG-6 promoter bind NF-IL6 with different efficiencies. A, an end-labeled oligonucleotide containing the NF-IL6* site was incubated without (lane 1) or with increasing amounts of recombinant GST-NF-IL6-3 (20, 60, and 100 ng, respectively, lanes 2-4). In the remaining groups a mixture of the labeled NF-IL6* oligonucleotide and 100 ng/ml NF-IL6-3 was incubated with one of the following: a 50-fold molar excess of the unlabeled 5` NF-IL6 oligonucleotide (lane 5), a 50-fold molar excess of unlabeled NF-IL6* oligonucleotide (lane 6), or antibody against NF-IL6 (lane 7). B, increasing amounts of GST-NF-IL6-3 (20, 60, and 100 ng, respectively, lanes 2-4 and 7-9) were incubated with the end-labeled 5` NF-IL6 or NF-IL6* oligonucleotide. Lanes 1 and 6 contain probes only. The binding of 100 ng NF-IL6-3 was competed for with a 50-fold molar excess of cold NF-IL6* oligonucleotide (lane 5). EMSA was performed as described under ``Experimental Procedures.'' The arrow marks the position of the protein-DNA complexes, separated from the free probe (FP).



Treatment with TNF and IL-1 Enhances Binding of Nuclear Proteins to the NF-IL6* Site

Earlier we showed that treatment of FS-4 cells with TNF or IL-1 increased nuclear protein binding to the AP-1 site in the TSG-6 promoter(4) . To determine if protein binding to the NF-IL6 sites is also regulated by TNF and IL-1, we prepared nuclear proteins from TNF- or IL-1-treated FS-4 cells and analyzed their binding to both NF-IL6 sites. Nuclei from unstimulated cells contained a small amount of protein that bound to the NF-IL6* probe (Fig. 6, lane 2), but no constitutive binding to the 5` NF-IL6 oligonucleotide was detected (lane 9). Treatment of cells with either TNF or IL-1 increased the binding of nuclear protein to NF-IL6* (lanes 3 and 4) as well as to the 5` NF-IL6 site (lanes 10 and 11). Formation of protein-NF-IL6* complexes was inhibited by competition with a molar excess of the unlabeled 5` NF-IL6 oligonucleotide (lane 5) and cold NF-IL6* oligonucleotide (lane 6). In contrast, competition with the unlabeled AP-1 oligonucleotide did not interfere with formation of the protein-DNA complex (lane 7). These results suggest that TNF and IL-1 enhance NF-IL6 binding to the NF-IL6* site and, though less efficiently, also to the 5` NF-IL6 site.


Figure 6: TNF and IL-1 enhance binding of nuclear proteins to both NF-IL6 sites. Nuclear proteins were prepared from unstimulated FS-4 cells (lanes 2 and 9) or from cells that were stimulated for 2 h with TNF (lanes 3 and 10) or IL-1 (lanes 4-7 and 11). Binding of nuclear proteins (4 µg) to the end-labeled NF-IL6* oligonucleotide (lanes 1-7) or 5` NF-IL6 oligonucleotide (lanes 8-11) was analyzed in EMSA. Nuclear extracts were preincubated with a 50times molar excess of unlabeled 5` NF-IL6 oligonucleotide (lane 5), unlabeled NF-IL6* oligonucleotide (lane 6), or with an unlabeled oligonucleotide containing the AP-1 site from the TSG-6 gene (lane 7). Lanes 1 and 8 contain probes only. The protein-DNA complex, separated from the free probe, is marked by an arrow.



Mutation in the NF-IL6* Site Abolishes NF-IL6 Binding

Site-directed mutagenesis was used to further evaluate the binding of NF-IL6 protein to the two NF-IL6 sites in the TSG-6 promoter. Probes with both NF-IL6 sites unaltered and probes with mutations introduced into one of the two NF-IL6 sites were used in EMSA. Complexes C1, C2, and C3 were formed with a probe from the wild type TSG-6 promoter (Fig. 7). A significant reduction in the formation of all three complexes was seen with a probe that contained a mutated promoter-distal NF-IL6 site (NF-IL6 M). The formation of all three complexes was abolished in EMSA with a probe containing a mutated NF-IL6* site (NF-IL6* M), despite the presence of the unaltered 5` NF-IL6 site. These results suggest cooperative binding of NF-IL6 by the two NF-IL6 elements and show that the NF-IL6* site is essential for NF-IL6 binding.


Figure 7: Mutation of NF-IL6* site abolishes binding of NF-IL6 protein to the TSG-6 promoter. The end-labeled HindIII-ScaI fragment, isolated from the wild-type (WT) TSG-6 promoter or from the promoter with a mutated 5` NF-IL6 site (NF-IL6 M) or mutated NF-IL6* site (NF-IL6* M) was incubated in the absence(-) or in the presence (+) of GST-NF-IL6-3 protein (10 ng). NF-IL6 M and NF-IL6* M probes were double-stranded versions of the oligonucleotides shown in Fig. 1. EMSA was performed as described under ``Experimental Procedures.'' Arrows mark complexes C1, C2, and C3, separated from the free probe (FP).



Both NF-IL6 Sites Participate in Transactivation by NF-IL6

Having shown that both NF-IL6 sites bind NF-IL6, albeit with different efficiencies, we examined the relative importance of the two sites for transactivation of the TSG-6 promoter by NF-IL6. HeLa cells were cotransfected with the NF-IL6 expression vector (pCMV-NF-IL6) together with a CAT reporter gene linked to one of the following: the wild-type TSG-6 promoter (pBBCAT, WT), the same region containing a mutated 5` NF-IL6 site (NF-IL6 M), or a mutated NF-IL6* site (NF-IL6* M). Disruption of the 5` NF-IL6 site strongly reduced transactivation, and mutation in the NF-IL6* site virtually abolished transactivation by NF-IL6 (Fig. 8). These findings suggest that the two NF-IL6 sites are functionally interdependent and that both sites are needed for efficient transactivation by NF-IL6.


Figure 8: Both NF-IL6 sites are necessary for transactivation of the TSG-6 promoter by NF-IL6. Analysis of TSG-6 promoter activity in HeLa cells transfected with the pBBCAT reporter plasmids alone (control) or together with 1 or 5 µg of the NF-IL6 expression vector. The promoter constructs used were: the wild-type promoter (WT), the promoter with a mutated 5` NF-IL6 element (NF-IL6 M), or promoter with a mutated NF-IL6* site (NF-IL6* M). CAT assays were performed and analyzed as described under ``Experimental Procedures.'' Results representative of three independent experiments are shown. White bar, control; narrow striped bar, NF-IL6, 1 µg; wide striped bar, NF-IL6, 5 µg.




DISCUSSION

In earlier reports we described the cloning of the 5` regulatory region of the TSG-6 gene and identified some of the cis-acting elements necessary for its inducibility(3, 4) . Deletion analysis and site-directed mutagenesis showed that an AP-1 (-126 to -119) and an NF-IL6 (-106 to -114) site are important in TSG-6 gene activation by the proinflammatory cytokines TNF and IL-1 (4) . The integrity of both the AP-1 and NF-IL6 sites was essential for inducibility by TNF, whereas induction by IL-1 required the NF-IL6 site and was greatly enhanced by the AP-1 site. In the present study we identified a second NF-IL6 binding site in the TSG-6 promoter at positions -92 to -83, separated from the earlier-identified promoter-distal NF-IL6 site by only 13 base pairs. We show that the two NF-IL6 sites cooperate in NF-IL6 binding and that both sites are essential for transactivation of the TSG-6 promoter. Thus, there are at least three interacting elements that control the induced expression of the TSG-6 gene: two NF-IL6 sites and an AP-1 element.

Several other promoters are known to harbor two NF-IL6 sites. Activation of the IL-1beta gene promoter by lipopolysaccharide and TNF was shown to be mediated by two adjacent NF-IL6 elements; a mutation in either one of these sites did not interfere with the function of the other site, whereas a mutation in both sites abolished activation(21) . In the IL-6 gene promoter, two NF-IL6 sites exert a positive regulatory activity and can act independently, although the 5` NF-IL6 site was demonstrably less important than the 3` NF-IL6 site(20) . The IL-6 gene promoter retained significant transcriptional activity even if mutations were introduced into both NF-IL6 sites; this residual activity was shown to be mediated by a potent NF-kappaB element located downstream of the two NF-IL6 sites(20, 26) . In contrast, in the E1A-responsive adenovirus E2ae promoter, a promoter-proximal NF-IL6-responsive site functions as a dominant negative regulatory site, whereas a promoter-distal NF-IL6 recognition sequence is positively or negatively regulated by NF-IL6 depending on the composition of NF-IL6 isoforms(11) . Similarly, in the promoter of the acute phase reactant alpha1-acid glycoprotein, two NF-IL6 sites were shown to function in an antagonistic manner; the promoter-distal NF-IL6 site was inhibitory, whereas the proximal NF-IL6 element was stimulatory in response to a combined activation with transfected glucocorticoid receptor and NF-IL6(17) . Multiple binding sites for NF-IL6 are also found in the long terminal repeat of human immunodeficiency virus, type 1(27) , and in the promoters of the genes encoding human haptoglobin(28) , human C-reactive protein(29) , and the third component of complement(30) , but the functional interplay of these NF-IL6 motifs has not been analyzed. Thus, the TSG-6 promoter is unique among the promoters known to contain two NF-IL6 sites in that mutation of either site virtually abolished activation (Fig. 8).

TSG-6 is a single copy gene, located on human chromosome 2(3) . Although its exact function is not yet known, high concentrations of TSG-6 protein are found in the joint fluids of arthritis patients, and synovial cells from arthritic joints synthesize TSG-6 mRNA and protein (2) . In contrast, TSG-6 protein was not detectable in the synovial fluids of individuals with no known history of arthritis. Elevated TSG-6 protein was also found in sera of patients with bacterial sepsis and of human volunteers injected with lipopolysaccharide (2) . (^3)A role for TSG-6 in inflammation and the acute phase response is also supported by the recent demonstration that TSG-6 protein forms a stable complex with inter-alpha-inhibitor, a serine protease inhibitor present in normal plasma(32) . TSG-6 expression is tightly regulated, as judged by the absence of detectable constitutive expression in normal human FS-4 fibroblasts and most other types of cells examined(1, 2, 3, 19, 31) . However, high concentrations of TSG-6 protein are produced upon appropriate stimulation. Thus, the design of the TSG-6 promoter must be such that little or no TSG-6 expression takes place in unstimulated cells, whereas a high level of synthesis can be rapidly reached during inflammation. The presence of two interdependent positively acting NF-IL6 sites appears to fulfill these requirements for tight repression and a rapid, efficient upregulation of synthesis.

Another important feature of the TSG-6 promoter is the presence of an AP-1 site, separated from the 5` NF-IL6 site by only 4 base pairs (Fig. 4B). Earlier we showed that a mutation in the AP-1 site (or its deletion) abolished the response to TNF and significantly decreased inducibility by IL-1(4) . Transcriptional activation via the AP-1 site is likely mediated by Fos/Jun proteins because treatment of cells with TNF or IL-1 increased nuclear protein binding to the AP-1 site of the TSG-6 promoter, and the formation of the complex was prevented by antibodies to Fos and Jun(4) . Yet, the AP-1 site is insufficient to mediate efficient transcriptional activation of the TSG-6 promoter because a mutation in any one of the two NF-IL6 elements virtually abolished promoter activation by NF-IL6, despite the presence of the intact AP-1 site (Fig. 8). The AP-1 site may also play a role in the negative regulation of TSG-6 gene expression. We have shown that the inhibitor isoform of NF-IL6 can bind directly to the AP-1 site in the TSG-6 gene and, thereby, at high concentration, repress transcriptional activation(4) . In addition, NF-IL6 protein can form heterodimeric complexes with Fos or Jun through their respective basic leucine zipper regions, resulting in a repression of transcriptional activation by NF-IL6(7) . Whether the latter mechanism operates in the negative regulation of the TSG-6 promoter has not been directly demonstrated. Thus, a striking feature of the TSG-6 promoter is the close proximity and interdependence of its three major enhancer elements: two NF-IL6 sites and an AP-1 motif.


FOOTNOTES

*
This work was supported by National Cancer Institute Grant R35-CA49731 (to J. V.) and by American Cancer Society Grant IM548 (to S. C.-K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY, 10021.

To whom correspondence should be addressed: Dept. of Microbiology, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-5315; Fax: 212-263-7933.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; TSG-6, TNF-stimulated gene 6; IL-1, interleukin 1; NF-IL6, nuclear factor for the IL-6 gene; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase.

(^2)
W. Hsu, T. K. Kerppola, T. Curran, and S. Chen-Kiang, manuscript in preparation.

(^3)
H.-G. Wisniewski, A. F. Suffredini, and J. Vilcek, unpublished observations.


ACKNOWLEDGEMENTS

We thank Wei Hsu for providing recombinant NF-IL6 protein, Paul Schwenger and Anne Altmeyer for helpful discussions, Angel Feliciano for technical assistance, and Ilene Totillo for preparation of the manuscript.


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