(Received for publication, September 1, 1994; and in revised form, December 7, 1994)
From the
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.
Tumor necrosis factor (TNF)-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/EBP, 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-
B(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. (
)
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) .
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.
Figure 2: Oligonucleotides used in the electrophoretic mobility shift assay. Arrows indicate the orientation of the NF-IL6 binding sites.
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.
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).
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 50 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.
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).
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.
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-1 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-
B 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
1-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) . ()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-
-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.