(Received for publication, October 27, 1995; and in revised form, January 3, 1996)
From the
The NF-B and NF-IL6 elements have previously been shown to
play an important role in regulation of both the mouse and human
interleukin-6 gene. Between these two elements lies a G/C-rich
sequence, which contains three repeats of the element CCACC, protein
binding to which has not been previously characterized. In this study
we demonstrate that the transcription factor Sp1 binds to these repeats
and plays an important role in basal and in inducible expression of the
murine interleukin-6 gene.
IL-6 ()is a cytokine with pleiomorphic biologic
effects expressed in fibroblasts, monocytes/macrophages, endothelium,
keratinocytes, and other mesenchymal and epithelial cells in response
to a variety of noxious stimuli, including tumor necrosis factor-
,
IL-1
, LPS, platelet-derived growth factor, and
interferons(1) . It is also produced constitutively in some
lymphomas, sarcomas, and carcinomas and is elevated in patients with
systemic bacterial infections (in patients with cardiac myxoma,
rheumatoid arthritis, Castleman's disease, monoclonal
gammopathies, psoriasis, septic shock, and
AIDS(2, 3) ).
Isolation and analysis of human (4, 5, 6) and mouse (7) genomic IL-6
clones have revealed a highly similar structure of regulatory elements.
These consist of a c-fos serum response-like element (8) shown to be the binding site for NF-IL6(9) , a cAMP
response element binding protein site just upstream of the NF-IL6 site,
and an NF-B
site(4, 5, 6, 7, 8, 10, 11) .
At a more upstream location are found putative multiresponse,
glucocorticoid response, and ETS-responsive elements (12) .
Tanabe et al.(7) and Ray et al.(8) first noticed a GT-rich region lying between the
NF-
B and NF-IL6 sites, which they felt may have homology to the fos retinoblastoma control element. However, no specific
deletions or characterization of factor binding to this region were
performed. Since then, there has been little further work on this
region.
Studies with a wide variety of inducers of IL-6 (LPS,
poly(IC), phytohemagglutinin, and phorbol 12-myristate 13-acetate) have
shown that induction correlates strongly with loading of NF-B and
that binding to this element in the IL-6 promoter is necessary for
response to these agents(11) . IL-1 and tumor necrosis factor (13) are probably the most potent cytokine up-modulators of
IL-6 secretion in vivo and may be synergistic(14) .
Functionally significant up-modulation by IL-1 and tumor necrosis
factor correlates with increased binding at the NF-IL6 site (15, 16, 17) and changes at the NF-
B
site(18, 19) .
We have previously demonstrated that
mice expressing the HTLV-I tax gene develop fibroblastic
tumors(20) . These tumors show constitutive translocation of
the p50 and p65 subunits of NF-B and constitutive activation of
the IL-6 gene. Both IL-6 expression and tumorigenesis are highly
dependent on NF-
B in this system since antisense inhibition of p65
profoundly affects both. By other approaches, we have demonstrated that
IL-6 expression is an important autocrine growth factor for these
transformed fibroblasts(21) . In order to better understand
transcriptional regulation of IL-6 we have performed promoter mapping
and mutagenesis of the region immediately upstream of the NF-
B
site. Here, we have identified Sp1 as prominently binding in this
upstream region, which is functionally important in transcriptional
regulation. Sp1 could serve as an important bridge in binding between
NF-
B and C/EBP isoforms in the IL-6 promoter.
B line cells were grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Antisense p50 and p65 treatments were as described previously(23) . Treatments were at 40 µg/ml for 48 h.
Figure 1:
A, in vivo footprinting of the
IL6 regulatory region from -38 to -300 in fibroblast (Fib) tumor cell line B. Naked (NAK) DNA was treated
with 2.5 units of DNase1/ml in vitro; (all subsequent in
vivo digestions were performed with 5 units of DNase1/ml).
Untreated (Untr) cells and cells treated with 40 µg/ml
NF-Bp65 antisense (p65-AS), NF-
Bp65 sense (p65-S), NF-
Bp50 antisense (p50-AS), or
NF-
Bp50 sense (p50-S) for 3 days are shown. Locations of
observed in vivo footprints are indicated by bars on
the right side of each set of samples. Locations of putative
transcription factor binding sites are indicated on the left. AP1, activator protein 1; IRE, interferon response
element; IEC, interferon enhancer core; NFIL6,
nuclear factor interleukin 6; CRE, cAMP-responsive element; CAAT, CCAAT box(7, 55) ; NF
B,
nuclear factor-
B(10) ; OCT, similar to SV40
octamer sequence(30) . B, diagram of target sequences.
At the top is shown a comparison of mouse and human IL-6
proximal promoters (numbering with respect to major start of
transcription). Gel shift probes and competitors used in this study are
shown below. All probes were used as
duplexes.
For LPS stimulation, cells were transfected with plasmids for 5 h in the absence of serum. The supernatant was then replaced by complete media containing 1 µg/ml LPS (Sigma, Escherichia coli), followed by incubation for an additional 18 h. DNA:Lipofectamine ratios were 1:8 (µg/µg).
We
have previously described the effects of treatment of B-line
fibroblasts with antisense oligonucleotides directed against the p50 or
p65 subunits of NF-B. Expression of IL-6 in particular is
profoundly inhibited(22, 23) . Removal of NF-
B
from the IL-6 locus by treatment with p50 or p65 antisense abolished
IL-6 in vivo footprints in the NF-
B response element (Fig. 1A). In addition, antisense treatment also
affected adjacent footprints extending as far upstream as the NF-IL6
elements (Fig. 1A). Sense treatments had no effect, and
there was no effect of either NF-
Bp65 or NF-
Bp50 antisense
treatment on footprints overlying the human T cell leukemia virus-I
long terminal repeat promoter-driving tax in these cells (24) (data not shown). Therefore, the effects of NF-
B
antisense treatment appear specific for loci that bind NF-
B. These
would include the promoters of many genes other than IL-6. These data
may suggest cooperativity of binding of transcription factors in the
regions between the NF-
B and NF-IL6 sites.
Somewhat surprising
was an extremely prominent footprint over the C-rich region just
upstream of the NF-B site. As noted in the Introduction, this has
not previously been appreciated as a strong site of transcription
factor binding (see (11) ). It consisted of three repeats of
the pentanucleotide sequence CCACC, similar to sequences previously
shown to bind zinc finger proteins of the type C2H2. The boundaries of
these regions are shown schematically in Fig. 1B. The
doubly underlined regions highlight the close proximity of the NF-IL6,
CCACC, and NF-
B motifs. This region is highly conserved between
mouse and human.
Figure 2:
Gel shift analysis of CCACC complex. A, general characteristics. Comparisons of extracts obtained
from Balb 3T3 (lanes 1, 2, 7, and 8) show no
significant differences from that in the B line (lanes
3-6). The activity is predominantly nuclear (Nuc)
with little in the cytoplasm (Cyto). The complex is not
induced by phorbal plus ionomycin treatment (PMA/CaI). The
complex is competed well by itself (lane 14) but not by
NF-B sequences obtained from the Ig or IL-6 locus (see Fig. 1B). We have previously shown that a single base
pair mutation at G (Mut Ig-
B) completely blocks NF-
B
binding(23) . Untr, untreated. B,
phosphocellulose fractionation. BNE, B line nuclear extract.
Protein concentrations of phosphocellulose column KCl gradient eluates
were measured by the Bradford assay (lower panel). The binding
activities of each fraction were screened by EMSA using CCACC wild type
probe (upper panel). The two bands are indicated by arrows. C, affinity purification. Protein
concentrations of CCACC affinity column KCl gradient eluates (lower
panel) and its binding activity (upper panel) were
detected by EMSA. Phosphocellulose column 600 mM KCl eluted
fraction was pooled and loaded onto the affinity column. PS,
phosphocellulose column. D, Southwestern blotting with the
CCACC probe. The amount of protein loaded from each step is as follows:
20 µg of B nuclear extract (lane 1), 10 µg of
phosphocellulose column flow-through (lane 2), 2 µg of
each phosphocellulose-eluted fraction (lanes 3-9), 0.2
µg of affinity column-purified 400 mM KCl fraction (lane 10). E, Western blot using anti-Sp1 Ab is
shown. The amount of protein loaded is as same as in D. PC, phosphocellulose; AF, affinity
purified.
One protein, running at approximately 105 kDa on the Southwestern blot, was strongly enriched by both phosphocellulose and affinity fractionation (lanes 6 and 10). Based on the apparent size and target sequence, this protein was most consistent with an Sp1 family member. To further evaluate this, fractionated extracts were analyzed by Western blotting using a commercially available Sp1 antibody. The results are shown in Fig. 2E (lanes 4 and 5). Sp1 activity was shown to be highly enriched in these same fractions, further suggesting that the purified factor was indeed Sp1.
To confirm Sp1 binding activity, gel shift competitions were
performed with probes conforming to known consensus sequences. When
analyzed at high resolution, the CCACC binding complex could be
resolved as two separate closely spaced bands (Fig. 3, A and B). Both bands were competed efficiently by native
probe. Competition with the classic SV40 Sp1 probe (sequence shown in Fig. 1B) competed only the upper band (Fig. 3A), whereas another GC-rich target, AP2
(sequence shown in Fig. 1B), only partially competed
the lower band and only at very high molar ratios (Fig. 3A). The AP1 probe was unable to compete. This
also suggested that the upper band contained an Sp family member. To
identify this member, an Sp1 specific antibody was used in supershift
analysis (Fig. 3B). This was specifically able to shift
the upper band, confirming the upper band as Sp1. Control NF-B and
cAMP response element binding protein antibodies had no effect on
either band (data not shown).
Figure 3: EMSA analysis using CCACC wild type probe. A, competition was done with cold CCACC probe itself, Sp1, AP1, and AP2 probes. Unlabeled CCACC could completely abolish the complexes, but the SV40 Sp1 could only compete the upper complex. AP2 had a partial effect, and AP1 did not compete any of the complexes. B, supershift assay with anti-Sp1 antibody. The upper complex was supershifted as indicated by the arrow.
Figure 4: EMSA analysis using CCACC wild and mutant probes. The upper CCACC complexes (indicated by arrow) are completely abolished using the CCACC mutant (mut) as probe. Cross-competitions were performed at 50 molar excess.
To analyze functional consequences of Sp1 binding to
the CCACC region, a luciferase vector driven by the -179 to
+34 region of the murine IL-6 promoter was constructed by PCR.
This promoter therefore contained the NF-IL6, CCACC, and NF-B
elements as well as the native IL-6 TATA box. The mutant CCACC region
was introduced by directional PCR cloning. Authentic sequences of both
the wild-type and mutant promoter constructs were confirmed by
automated sequencing.
The promoters were analyzed by transfection
into both unstimulated and LPS-stimulated 3T3 fibroblasts. We have
previously shown that Balb/c3T3 cells express low levels of nuclear
NF-B and secrete minimal levels of IL-6 constitutively, but both
NF-
B and IL-6 are highly inducible by LPS within 6 h. The results
of these transfections are shown in Fig. 5. In unstimulated 3T3
cells, basal levels of luciferase for the wild type promoter were
roughly 4 times that for the mutant at 24 h (Fig. 5B),
increasing to 23 times when assayed at 48 h (Fig. 5A).
This held true over a 3-fold range of plasmid dose. This difference was
highly significant (p = 0.0062 (F(1,2)
= 10610) by analysis of variance). Previous studies have
demonstrated little induction in transient chloramphenicol
acetyltransferase assays using LPS when assayed at 48 h(8) . By
performing these assays at 24 h and using the sensitive luciferase
activity as a marker, we were able to demonstrate reproducible 2.6-fold
induction of the wild type plasmid, which was significant (p < 0.05; t = 4.5), but only 1.5-fold induction
in the mutant (Fig. 5B). Identical results were
obtained with two different plasmid preps.
Figure 5: Luciferase assays. Comparison of wild type and CCACC mutant IL-6 luciferase vectors by transient transfection in Balb/c3T3 cells. A, 48-h transient transfection assay in which the two plasmids are compared at doses ranging between 2.5 and 7.5 µg (error brackets for mutant plasmid are too small to see). B, LPS induction of both plasmids. Transient transfections were assayed at 24 h to optimize LPS response.
Taken together, these data show that Sp1 binding is important for both basal and inducible IL-6 expression.
Our in vivo footprinting studies suggested that the
CCACC-containing region was constitutively occupied by transcription
factors in expressing fibroblasts. This region consists of three
repeats of the sequence CCACC. A promoter region with similar structure
has also been identified in the human eosinophil peroxidase gene (34) and the c-fos promoter(35) . Related
sequences had been previously demonstrated to bind a variety of C2H2
type zinc finger-containing transcription factors, which include THP-1,
GLI, ht, Zif 268 (EGR1), EKLF, Sp1-Sp4, and
YY1(36, 37, 38, 39) . Other proteins
with similar binding targets also include
Puf(40, 41) , AP2(42) , and
H4TF-1(43) . Supershift, Southwestern, and affinity
purification analyses suggest that the upper complex is Sp1. The
identity of the lower complex remains to be determined. Luciferase
experiments suggest that the binding of this complex is functionally
very important in both basal and induced IL-6 regulation.
Both p50
and p65 antisense inhibition of NF-B caused extensive and specific
change to the footprint between the NF-IL6 and NF-
B sites.
However, changes did not extend upstream of the NF-IL6 site. Ablation
of the footprints in this chromatin domain could occur through numerous
mechanisms. However, we feel it is most consistent with strongly
cooperative binding of transcription factors bound by these elements.
Cooperativity between NF-
B and NF-IL6 components has previously
been
demonstrated(44, 45, 46, 47, 48) .
Further, interactions between both the p50 and p65 subunits of
NF-
B may have important interactions with NF-IL6 for at least two
other genes(47, 48) . Positive cooperativity between
Sp1 and NF-
B has also been demonstrated to play an important role
in regulation of the human immunodeficiency virus
promoter(49) . Interactions between Sp1 and NF-IL6 have not
been previously described, and definition may have to await further
mutagenic experiments. In the case of mouse IL-6, a variety of family
members (C/EBP-
, -
, -
, and -
) may bind the NF-IL6
elements at various stages of the inflammatory
response(19, 50, 51) . In contrast, splicing
variants of NF-IL6 may play a similar role in human cells(52) .
Similarly, a variety of NF-
B family members may bind the IL-6
NF-
B element during immune maturation.
In the case of the IL-6 promoter, Sp1 may provide the scaffolding to facilitate these interactions. Indeed, Stein and co-workers(53, 54) have shown that Sp1 may participate in matrix attachment sites. Such attachment sites could ensure poising of the IL-6 promoter for rapid response to inflammatory stimuli.