(Received for publication, September 14, 1995; and in revised form, February 5, 1996)
From the
Interleukin-6 (IL-6) is the major cytokine inducing
transcription of human C-reactive protein (CRP) during the acute phase
response. STAT (signal transducers and activators of transcription)
family members, recently shown to be important mediators of the effects
of many cytokines including IL-6, generally induce their effects by
binding to palindromic sequences with TT(N)AA motifs. We
report an IL-6 responsive element in the proximal region of the human
CRP 5`-flanking region that bears a TT(N)
AA motif, which we
have termed CRP acute phase response element (CRP-APRE). In Hep3B
cells, IL-6 but not interferon-
was capable of activating CAT
constructs driven by the CRP promoter containing CRP-APRE.
Overexpressed STAT3 was able to transactivate CRP-chloramphenicol
acetyltransferase constructs through the CRP-APRE and was able to
enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or
an antigenically related molecule) bound to the CRP-APRE in response to
IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of
inducing expression of a construct consisting of the CRP-APRE and a
minimal thymidine kinase promoter lacking a C/EBP site. Taken together,
these findings indicate that STAT3 participates in the transcriptional
activation of CRP in response to IL-6.
A large number of systemic and metabolic changes, collectively
referred to as the acute phase response (APR), ()begin to
occur within hours after an inflammatory
stimulus(1, 2, 3) . Among these changes is a
reprogramming of the pattern of plasma protein gene expression in
hepatocytes, with consequent changes in blood concentrations of these
proteins. C-reactive protein (CRP) is a major acute phase protein in
humans, its concentration increasing more than 1000-fold in severe
inflammatory states.
Interleukin-6 (IL-6) appears to be the
principal regulator of most acute phase
proteins(3, 4) , although other
inflammation-associated cytokines also contribute to this process.
IL-1 and tumor necrosis factor-
have been found to
participate in induction of a broad subset of acute phase proteins, and
both transforming growth factor-
and
-interferon can induce
limited subsets of acute phase proteins (2) . IL-6 has been
shown to activate members of the C/EBP family of transcription factors
in hepatoma cell lines(5) . It has recently been found that
STAT (signal transducers and activators of transcription) family
members may also play a major role in mediating IL-6 effects (6, 7, 8, 9) . The binding of IL-6
to its receptor complex leads to phosphorylation of Janus kinase
kinases, with subsequent rapid (15-60 min) phosphorylation,
dimerization, and nuclear translocation of a transcription factor
originally named acute phase response factor (8) and since
designated STAT3(6) . STAT3 then binds to specific response
elements in the promoter regions of cytokine responsive genes. The
promoter regions of a number of human and rat acute phase genes contain
TT(N)
AA sequences (10, 11, 12, 13) capable of binding
STAT proteins. STAT1 (14) also appears to be activated by IL-6
as well as by IFN-
with similar rapid kinetics and binds to a
similar consensus motif called the
-interferon activation site
(GAS)(15, 16, 17) . Both STAT3 and STAT1 have
been found to be activated by a broad spectrum of cytokines and growth
factors, including IFN-
, epidermal growth factor, and IL-6-related
cytokines(17, 18) .
We have previously shown in the
human hepatoma cell line Hep3B that IL-6 activates transcription of
CRP, that IL-1, which has no effect alone, synergistically
enhances CRP transcription in the presence of IL-6, and that the
proximal 157 bp of the 5`-flanking region of CRP was sufficient to
confer IL-6 induction and IL-1
synergistic activation on
CRP-CAT(19) . Several cis-elements and trans-activators that
were required for CRP transcription in response to monocyte conditioned
medium and IL-6 have been characterized. Two C/EBP binding sites in the
proximal region of the CRP promoter have been shown to bind
IL-6-inducible C/EBP family members(20) , and two HNF-1 sites
have been shown to be adjacent to the C/EBP sites and to be necessary
for CRP transcription(21, 22) . Other regions
containing positive and negative cis-elements have also been found in
the 5`- and 3`-flanking regions of the CRP
gene(23, 24) .
We report here the finding of a
STAT3 response element in the human CRP promoter with the sequence
TTCCCGAA, which is necessary for optimal IL-6-induced transcription of
CRP. Oligonucleotides with the TT(N)AA motif have recently
been reported to specifically bind STAT3(25) . This finding
indicates that STAT3 or a closely related molecule participates in
mediation of the transcriptional effect of IL-6 on human CRP.
For cytokine induction, the
transfected cells were incubated with serum-free medium and exposed to
10 ng (100 units)/ml of IL-6, 10 ng (100 units)/ml IL-1, or 10 ng
(100 units)/ml IFN-
for up to 24 h. STAT3 stable cell lines were
generated by transfecting Hep3B cells with Rc/CMV-STAT3 by
electroporation as described above, followed by G418 (400 µg/ml)
selection. Single colonies were picked and expanded.
Figure 1:
Activation of
CRP-CAT 5` deletion constructs by IL-6 and IL-1 in Hep3B cells. A
diagrammatic representation of the CRP promoter (-157 to +3)
is shown at the top. The indicated C/EBP binding sites and
HNF-1 site have been described by others(20, 21) .
CRP-CAT constructs were transiently co-transfected with pRSV
-Gal
into Hep3B cells. Cells were treated with cytokines (IL-1
, 100
units/ml; IL-6, 100 units/ml) for 24 h. CAT activity (percentage of
butyrylated chloramphenicol) was measured as described under
``Experimental Procedures.'' CAT activities were normalized
by
-galactosidase activity to control for differences in
transfection efficiencies. The values are the averages of four
independent experiments performed in duplicate. The error bars indicate the standard errors.
The sequence disrupted in the B mutant, TTCCCGAA,
was found to be homologous to members of the family of acute phase
response elements (APREs) first identified in the rat 2M gene as
well as to the GAS (Table 2). It is noteworthy that the CRP
element has only four nucleotides between the conserved TT . . . AA,
although the surrounding sequences closely resemble
2M APRE.
Therefore, we named the element disrupted in mutant B CRP-APRE.
Figure 2:
Transactivation of
-157/+3CRP-CAT by STAT3 in response to IL-6 and IL-1.
-157/+3CRP-CAT (15 µg) was transiently co-transfected
with STAT3 expression plasmid Rc/CMV-STAT3 (2 µg) or STAT1
expression plasmid pMNC-91 (2 µg) plus pRSV-
GAL (5 µg). 2
µg of pRc/CMV vector DNA were employed where neither expression
plasmid was used. Cells were treated with cytokines (IL-1
, 100
units/ml; IL-6, 100 units/ml) for 24 h. CAT activity was determined and
normalized to
-galactosidase activity. Two independent experiments
with duplicate samples were performed, and similar patterns of
expression were obtained. CAT activity of the means of duplicate
samples in one of these experiments is
plotted.
Similar observations
were made for the endogenous CRP gene in Hep3B cells. In cells stably
transfected with a STAT3 expression construct, CRP responses to IL-6
and to the combination of IL-6 + IL-1 were markedly increased (Fig. 3).
Figure 3:
Northern analysis of endogenous CRP mRNA
accumulation in cells stably transfected with pRc/CMV-STAT3. Cells were
treated with IL-1 (100 units/ml), IL-6 (100 units/ml), and
[IL-1+IL-6] (100 units/ml, 100 units/ml) for 24 h.
RNA was loaded at 15 µg/lane. pCRP5 (for CRP) was used as a probe.
CRP signals are shown in the upper panel, and actin signals
are shown in the lower panel. The results from a single clone
of pRc/CMV-STAT3 stably transfected Hep3B cells are shown. The signal
in Hep3B treated with IL-6 only was visible with longer exposure (not
shown).
In contrast, overexpressed STAT1 was unable to
increase CAT expression of -123/+3CRP-CAT even in the
presence of IFN-
(Fig. 4). It was of interest that
IFN-
as well as IL-6 was able to enhance transactivation of
-123/+3CRP-CAT by STAT3 and that the combination of IL-6 and
IFN-
had some additive effect in the presence of overexpressed
STAT3.
Figure 4:
Transactivation of
-123/+3CRP-CAT by STAT3 but not STAT1 in response to
IL-6 and IFN-
. -123/+3CRP-CAT (15 µg) was
transiently co-transfected with STAT3 expression plasmid Rc/CMV-STAT3
(2 µg) or STAT1
expression plasmid pMNC-91 (2 µg) plus
pRSV-
GAL (5 µg). Cells were treated with cytokines (IL-6, 100
units/ml; IFN-
, 100 units/ml) for 24 h. CAT activity was
determined and normalized to
-galactosidase activity. Two
independent experiments with duplicate samples were performed, and
similar patterns of expression were obtained. CAT activity of the means
of duplicate samples in one of these experiments is
plotted.
To verify the hypothesis that STAT3 transactivation was exerted through CRP-APRE, the four block mutants of -123/+3CRP-CAT were employed in co-transfection experiments (Fig. 5). As expected, the response to cytokines in cells overexpressing STAT3 was greatly abolished in mutant B, while other mutants were only moderately affected. In another approach, we employed an antisense STAT3 expression construct in which the direction of the cDNA was reversed in the expression vector in the co-transfection experiments and found that antisense STAT3 was able to suppress IL-6 induction of CRP-CAT expression to one-third of vector-transfection control levels (data not shown).
Figure 5:
Transactivation of
-123/+3CRP-CAT and four block mutants by STAT3.
-123/+3CRP-CAT or mutant A, B, C, or D (15 µg) (see
``Experimental Procedures'') was transiently co-transfected
with STAT3 expression plasmid Rc/CMV-STAT3 (2 µg) and pRSV-GAL
(5 µg). Cells were treated with cytokines (IL-6, 100 units/ml;
IL-1
, 100 units/ml) for 24 h. CAT activity was determined and
normalized to
-galactosidase activity. Two independent experiments
with duplicate samples were performed, and similar patterns of
expression were obtained. CAT activity of the means of duplicate
samples in one of these experiments is
plotted.
Figure 6:
Identification of STAT3 bound to CRP-APRE
in response to IL-6. HepG2 cells were either untreated or treated with
IL-6 (100 units/ml) for 15 min. Nuclear extract (5 µg of protein)
as described under ``Experimental Procedures'' was incubated
with about 200 fmol of P-labeled CRP-APRE oligonucleotide
and analyzed by EMSA. In competition experiments (lanes
5-10), 10
and 50
molar excess of cold
oligonucleotides were included in the binding reaction. In supershift
experiments, either 1 µg of C-20 anti-STAT3 antibody (Santa Cruz),
control rabbit IgG, or anti-STAT5 antibody (lanes 11-13,
respectively) were employed in the binding
reaction.
To examine the time course of the binding between STAT3 and CRP-APRE, Hep3B cells were treated with IL-6 for various lengths of time. EMSA studies (Fig. 7) revealed that IL-6-induced binding of STAT3 to CRP-APRE was present within 15 min of IL-6 induction and was still detectable 18 h later.
Figure 7:
Binding of STAT3 to CRP-APRE in response
to IL-6 exposure for varying times. Hep3B cells were either untreated
or treated with IL-6 (100 units/ml) for the indicated length of time.
Nuclear extract (5 µg of protein) was incubated with P-labeled CRP-APRE oligonucleotide and analyzed by EMSA.
In the competition experiments, 50
molar excess of cold
oligonucleotides were included in the binding reaction. 1 µg of
C-20 anti-STAT3 antibody (Santa Cruz) was used for
supershifts.
Figure 8:
Transactivation of pAPRE-TK(-77)-CAT
by STAT3. pAPRE-TK(-77)-CAT contains two copies of the CRP-APRE
inserted upstream of a 77-bp fragment of the herpes simplex virus
thymidine kinase promoter driving the CAT gene (see ``Experimental
Procedures''). The orientation of the two CRP-APREs was the same
as in the CRP promoter. pAPRE-TK(-77)-CAT (15 µg) or the
vector plasmid pTK(-77)-CAT (15 µg) was transiently
co-transfected by electroporation into Hep3B cells with STAT3
expression plasmid Rc/CMV-STAT3 (2 µg) plus pRSV-GAL (5
µg). 2 µg of pRc/CMV vector DNA were used when Rc/CMV-STAT3 was
absent. Cells were untreated or treated with IL-6 (100 units/ml) for 24
h. CAT activity was determined and normalized to
-galactosidase
activity. Two independent experiments with duplicate samples were
performed, and similar patterns of expression were obtained. CAT
activity of the means of duplicate samples in one of these experiments
is plotted.
Our major findings were that overexpressed STAT3 but not
STAT1 was able to transactivate CRP-CAT constructs in response to
IL-6 stimulation through a GAS-like sequence that we have termed
CRP-APRE, that overexpressed STAT3 activated the endogenous CRP gene in
response to IL-6, that CRP-APRE was able to bind STAT3 in an IL-6
inducible fashion, and that CRP-APRE, in the presence of overexpressed
STAT3, conferred IL-6 inducibility on a heterologous promoter lacking
C/EBP binding sites in Hep3B cells. Taken together, these findings
indicate that STAT3 participates in the transcriptional activation of
C-reactive protein in response to IL-6.
Although both STAT3 and
STAT1 have been shown to bind the same GAS-like elements and
activate transcription of a number of genes(9, 35) ,
this was not the case for CRP-APRE. Only STAT3 and not STAT1
transactivated CRP-CAT containing wild-type CRP-APRE; IL-6 markedly
enhanced this transactivation. IFN-
in the absence of STAT3
overexpression had no effect on either CRP-CAT constructs or on the
endogenous CRP gene (data not shown). Accordingly, we conclude that the
ability of IFN-
to enhance CAT expression that we observed in
STAT3-transfected cells was due to activation of overexpressed STAT3 in
Hep3B cells. Similarly, EMSA demonstrated that CRP-APRE bound to STAT3
in a IL-6-dependent manner. This selective response to IL-6 but not
IFN-
has not been reported for promoters containing other APREs,
including
2M APRE(36) . STAT3 has been shown to be the
major STAT member mediating IL-6 signaling, whereas STAT1
is the
major STAT activated by IFN-
(17) . It is likely that the
selective binding of STAT3 determines the specific response of the CRP
gene to IL-6 and not to IFN-
.
This specificity of CRP-APRE for
STAT3 may be explained by its structure, TT(N)AA, which
differs from the TT(N)
AA motif found in other APREs. In a
recent study (25) in which the effects of the spacing between
the TT and AA core half-sites on the binding of the STAT complexes were
examined using synthetic oligonuleotides, it was found that
TT(N)
AA elements displayed general STAT binding, whereas in
contrast the TT(N)
AA motif bound only to complexes
containing STAT3. Interestingly, one of their synthetic
TT(N)
AA oligonucleotides matched the CRP-APRE defined in
this study. These results strongly support the conclusions of this
study. These authors also induced expression of a luciferase construct
containing four copies of TTCCCGAA in HepG2 cells with IL-6. This
result contrasts with our finding that expression of pAPRE-TK-CAT was
unaffected by IL-6 in Hep3B cells not transfected with STAT3. This
discrepancy could be due to the low level of activated STAT3 in the
Hep3B cell line or to the comparatively high copy numbers of TTCCCGAA
(four instead of two) in their constructs. To our knowledge, CRP-APRE
is the first example of a ``natural'' TT(N)
AA
cis-element shown to bind STAT3 in an IL-6-inducible manner, although
an element bearing the TTCCTGAC motif in the murine JunB gene was
reported recently to bind STAT3 (37) .
The binding of STAT3
to CRP-APRE in Hep3B cells was found to last at least 18 h, which
contrasts to the duration of 1 h reported in HepG2 cells with the
2M APRE(8) . This difference may arise from differences in
the cell lines used or the assay systems employed or may be due to
distinctive binding properties of each DNA oligonucleotide. Long term
activation of STAT proteins has been reported using GAS-like elements
such as pIRE of the interferon regulatory factor 1 gene in the human
breast carcinoma cell line T47D(38) . It is conceivable that in
our system, continuous activation of STAT3 contributes to the
``prolonged'' binding detected in EMSA. Alternatively,
inactivation of STAT3 signaling (e.g. dephosphorylation of
STAT3) may be slow in our cells.
Our findings suggest that the CRP-APRE may also participate in maintenance of basal expression of CRP. Mutation of CRP-APRE not only decreased IL-6 induction of CRP-CAT but also lowered the basal level of CAT expression. We observed additional IL-6-independent complexes not abolished by antibody against STAT3 in EMSA using CRP-APRE as a probe ( Fig. 6and 7). This finding raises the possibility that binding of an unidentified transcription factor(s) other than STAT3 may account for constitutive basal level expression of CRP-CAT.
The observations presented here
should not be taken to imply that STAT3 is the major transcription
factor participating in mediation of the CRP response to IL-6. The role
of C/EBP binding sites in the CRP response to IL-6 is well
documented(22) . Our finding that two copies of CRP-APRE in the
absence of overexpressed STAT3 were not sufficient to confer IL-6
responsiveness on a truncated minimal TK promoter, although subject to
other interpretations, raises the possibility that there is need for
one or more other response elements. The fact that a GAS-like IL-6
responsive element exists in close proximity to the two C/EBP binding
sites in the CRP promoter (and other IL-6 responsive promoters) points
to possible cooperative effects between STAT and C/EBP family members.
Relevant to this issue are our preliminary findings, in studies of
reporter constructs containing mutated C/EBP or CRP-APRE sites, which
suggest that these sites are cooperative rather than functionally
independent. The latter observation, if confirmed, would be consistent
with the abundant evidence supporting the fundamental role of
interactions between transcription factors in gene-specific
transcriptional regulation(39) . Among acute phase proteins,
for example, an element in the human hemopexin promoter has been shown
to bind a complex which contains STAT3 in response to
IL-6(40) ; the human hemopexin promoter, like CRP, also
demonstrates IL-6-inducible C/EBP binding(40) . Physical
interaction between C/EBP and NF-
B has been demonstrated in
numerous cases(41, 42, 43) . C/EBP
has
also been shown to physically interact and functionally synergize with
the glucocorticoid receptor in the induction of the rat
1 acid
glycoprotein promoter(44) . Whether C/EBP members have similar
interaction with STAT3 is under investigation. These examples indicate
how difficult it may prove to be to assign relative importance to
interacting transcription factors participating in the full CRP
response.
Similarly, our findings should not be taken to indicate
that other as yet unidentified transcription factors do not participate
in CRP induction by IL-6. Several studies indicate that unidentified
activity other than STAT3 may activate IL-6-induced
transcription(45, 46, 47) . Although STAT3
has been shown to bind to three CTGGGA elements in the -fibrinogen
gene(48) , a similar CTGGGA element in A
-fibrinogen was
reported to associate with an unidentified protein that was not
STAT3(47) . In another recent report, STAT3 was found to
contribute to but not to be sufficient to up-regulate specific IL-6
response element-containing reporter constructs(46) . It is
likely that various combinations of STAT3, C/EBP, and other as yet
undefined IL-6 responsive factors and elements in their unique promoter
contexts determine the activation mechanisms for each IL-6 responsive
acute phase gene. Our finding that CRP-APRE (2X) is not sufficient to
confer IL-6 responsiveness on the 105-bp TK promoter (which contains a
C/EBP site) raises the possibility that an optimal response may require
elements other than STAT and C/EBP, although other explanations for
these findings clearly exist.
Finally, it is premature to conclude,
as some have, that IL-6-induced expression of all acute phase response
genes requires STAT3(49) . Thus far, as indicated above, it is
known that both STAT and C/EBP family members can mediate IL-6-induced
gene transcription and that other unidentified proteins may have such
capabilities as well. Theoretically there may be genes whose response
to IL-6 is dependent on transcription factors other than STAT3. This
possibility is supported by our continuing studies of Hep3B cells
stably transfected with STAT3 in which CRP demonstrated an enhanced
response to IL-6 + IL-1 (Fig. 3). Preliminary studies
in these cells suggest that serum amyloid A does not display such a
response, implying that STAT3 does not play a substantial role in the
serum amyloid A response to these cytokines.
It should also be noted that the minimal elements required for inducible expression of the CRP gene in hepatoma cells are not sufficient to control expression of the human CRP gene in transgenic mice following LPS treatment(24) . The reasons for this discrepancy are unclear. One possibility is that the cytokine milieu induced in vivo by LPS may be more complex than the defined medium and defined cytokines used in our cell culture experiments. In addition, many genes are dependent for expression on the presence of distant regulatory elements (both positive and negative), which may be thousands of base pairs away. It should therefore not be surprising that findings of gene regulation employing relatively short DNA sequences cannot be replicated in vivo, where many more regulatory elements, both positive and negative, come into play.