(Received for publication, June 5, 1995; and in revised form, August 4, 1995)
From the
Fibrinogen, a hepatically derived class II acute phase protein,
is the product of three separate genes, (A, B
, and
).
The fibrinogen genes are expressed constitutively; however, their
transcription can be significantly up-regulated by interleukin-6 (IL-6)
and glucocorticoid. Inspection of the promoter region of the fibrinogen
gene revealed three hexanucleotide clusters of CTGGGA that are
recognized as class II IL-6 responsive elements. Functional analyses of
these regions (designated here as site I, site II, and site III
according to their position in the promoter) were performed using
luciferase reporter constructs and show a hierarchy of IL-6 response in
which site II was the preferred functional site, site I was the next
important site, and site III was the site least responsive to IL-6. Gel
mobility shift assays using 25-base pair oligonucleotide probes derived
from these three regions with the CTGGGA positioned in the middle and
nuclear extracts from IL-6-treated primary hepatocytes reveal the
presence of IL-6-induced high molecular weight complexes appearing 5
min after cytokine treatment. Supershift assays using anti-Stat3
antibody indicate that Stat3 is part of the IL-6-induced complex formed
on the three
chain probes. The binding of Stat3 to the IL-6
responsive elements of the
probes is significantly weaker than to
an
-macroglobulin probe. These findings show for the
first time that Stat3 is involved in associating with the IL-6
responsive elements of fibrinogen
chain, a class II acute phase
gene other than
-macroglobulin.
During an acute phase inflammatory response, the expression of a
specific subset of hepatic genes are positively or negatively regulated
by glucocorticoid and cytokines(1, 2, 3) .
These acute phase response (APR) ()genes are classified into
two groups based on their response to the inducing
cytokines(4, 5) . Class I genes are up-regulated by
both IL-1 and IL-6. The NF-IL6 (C-EBP
) binding site with a
T(T/G)NNGNAA(T/G) consensus sequence in the promoter region has been
identified as the cytokine responsive element for this group of
genes(6, 7, 8) . Class II genes are regulated
by IL-6 and related cytokines (IL-11, leukemia inhibitory factor,
oncostatin M, and ciliary neurotrophic factor) but not by
IL-1(4, 5) . Recently published results on the
-macroglobulin gene demonstrated that an acute phase
response factor (also named Stat3) was tyrosine-phosphorylated in
response to IL-6, translocated into the nuclei, and specifically
associated with a CTGG(G)AA consensus element in the promoter of the
-macroglobulin gene leading to an up-regulated
transcription of this
gene(9, 10, 11, 12, 13, 14) .
The fibrinogen molecule is composed of three pairs of polypeptide
chains, A, B
, and
, that form a dimer
(A
-B
-
)
. Each chain is encoded by a single
gene, and all three fibrinogen genes contain CTGG(G)AA consensus
regions in their promoters that were originally suggested to be the
cytokine responsive element(15) . In previously published
studies on the fibrinogen B
chain promoter, the importance of the
CTGG(G)AA region was demonstrated using reporter gene functional
assays. Unfortunately, no information was presented in these studies on
the DNA-binding protein that associates with the CTGGGA
region(16, 17, 18) . Recently reported
studies of the fibrinogen A
chain promoter showed that a 50-kDa
protein specifically bound to the CTGGGA region in this gene that
responds to the IL-6 signal(19) . These findings were somewhat
unexpected because a similar CTGGGAA sequence in the
-macroglobulin gene had been used to identify a new
IL-6-induced transcription factor Stat3 (or APR
factor)(13, 14) . To date there are no published
results showing the IL-6-induced protein complexes binding on the
CTGG(G)AA elements of fibrinogen gene promoters. Furthermore, it has
not been established that Stat3 acts as a signal transducing regulation
of all class II APR genes.
For the fibrinogen gene, it has
been shown that there are three important elements for the basal level
transcriptional regulation, an Sp1 site, a CAAT enhancer-binding
protein binding site, and an SV40 major later promoter transcription
factor binding site(20, 21) . The IL-6 responsive
element of the
gene has not yet been clearly identified. The
fibrinogen
chain 5` promoter region contains three CTGG(G)AA
sites, which can be considered as the putative IL-6 responsive
elements. In the studies presented here, the three potential IL-6
responsive regions were examined by functional analysis and gel shift
assay. Data from functional studies show that the site II CTGGGAA
region is the major IL-6 responsive element of fibrinogen
chain
gene and that site I is the next important element; all three CTGG(G)AA
regions are necessary for achieving maximal response. The gel mobility
shift assays reveal that IL-6-induced protein complexes form on each of
the CTGGGAA elements, and unlike that of the A
gene, Stat3 is part
of these complexes. Furthermore, the complexes are NEM-sensitive and
contain the phosphorylated tyrosine residues.
Figure 1:
DNA sequence
review. A partial DNA sequence (-341 to +9 bp) of the
fibrinogen chain promoter region shows the defined SV40 major
later promoter transcription factor, CAAT enhancer-binding protein, Sp1
binding sites (which are underlined)(15, 20, 21) , the three
putative IL-6 responsive CTGG(G)AA regions (which are in boxes), the TATA box, and the transcription initiation site (+1).
The initial step to identify the functional
IL-6 responsive elements on the fibrinogen chain promoter was
performed by deletion mapping. Different lengths of
chain
promoter DNA fragments were obtained by polymerase chain reaction using
different 5` primers and subcloned into a luciferase reporter gene
vector pXP2 for functional assay (24) (Fig. 2). The
results show that the isolated fibrinogen
chain promoter
fragments (-1540 to +54 bp) can introduce the IL-6 response
to the downstream luciferase gene and the IL-6 response can be measured
directly by comparing the luciferase activity. The IL-6 response
decreased significantly when the
chain promoter 5` region was
deleted from -300 (F3-pXP2) to -74 bp (F4-pXP2), indicating
the
chain promoter region from -300 to +54 contains
the important region(s) for the IL-6 response.
Figure 2:
5` deletion mapping of fibrinogen
chain promoter. Different constructs were made by subcloning the DNA
fragments of different lengths of fibrinogen
chain promoter
regions into the promoterless luciferase reporter gene vector pXP2. All
constructs were transfected into H35 cells, and IL-6 response was
tested as described under ``Experimental Procedures.'' The
graphic shows the mean value of four individual experiments, and the error bars indicate the standard
deviation.
Within the -300
to +54 bp region of the chain promoter, three separate
CTGG(G)AA regions are located. To determine the contribution of each
CTGG(G)AA region in the IL-6 response, selective mutation functional
analyses were performed. The fragment containing -300 to +54
bp was subcloned into the luciferase reporter gene vector pGL2
(Promega), and site-directed mutagenesis was carried out as described
above. The selective mutated sites were confirmed by DNA sequencing.
The mutagenized constructs were transfected into H35 cells for
functional analysis. Results shown in Fig. 3indicate that the
site II CTGGGAA region (-146 to -140) on the fibrinogen
chain promoter exerted the major response to the IL-6 signal.
Either complete deletion or specific mutation of this region
significantly reduced the IL-6 response, as shown by constructs M1, M5,
M8, M9, and N2. The site I and site III CTGGAA regions are also
important to obtain maximal IL-6 response, because deletion of or
mutation within these regions also affected the IL-6 response, shown in
constructs M4, N1, and M7. These results indicate that all three sites
are required for achieving a full IL-6 response, and the comparison of
the constructs F3, M1, M4, and N1 indicate that site II is the most
important IL-6RE, site I is next, and site III is the least significant
IL-6RE. The constructs M9 and M10, which deleted all three CTGG(G)AA
regions, show no response to IL-6, which verified again that the
CTGG(G)A regions on the
chain promoter are essential for the IL-6
response.
Figure 3:
Functional analysis of the three CTGG(G)A
regions on the fibrinogen chain promoter. Different constructs
were made by subcloning the
chain promoter region (-300 to
+54 bp) into the basic luciferase reporter gene vector pGL2
(Promega) and selectively mutating or deleting the three CTGG(G)AA
regions. All the constructs were transfected into the H35 cells, and
IL-6 response was tested as described under ``Experimental
Procedures.'' The graphic shows the mean value of four individual
experiments, and the error bars indicate the standard
deviation. For selective mutation, the site II CTGGGA region was
changed to AGATCT, and the site III CTGGGA region was changed to
AGTTCC, which are indicated as
in the
drawing.
Figure 4:
Identification of the IL-6-induced
complexes associated with the chain CTGG(G)A regions by
electrophoretic mobility shift assay. A, electrophoretic
mobility shift assay was carried out as described under
``Experimental Procedures.'' The oligonucleotide probes used
for gel shift assays are shown in Table 1. Lanes
1-3,
MG,
-macroglobulin gene probe; lanes 4-6,
chain site I probe; lanes 7-9,
chain site II
probe; lanes 10-12,
chain site III probe.
Different nuclear protein extracts used for gel shift assays were
isolated from different time IL-6-treated primary hepatocyte, indicated
as 0, 2, or 5 min (0, 2`, and 5`,
respectively). The arrows on the left side indicate
the IL-6-induced complexes formed on the
-macroglobulin gene probe (from top to bottom, the complex contains Stat3-p97-p46, homodimerized
Stat3, heterodimerized Stat3 and Stat91, and homodimerized Stat91,
according to the published literature(26) ). The arrow on right side indicates the IL-6-induced complexes formed
on the
chain probes. The arrow that is labeled A indicates the unique complex formed on the site II probe, and the arrow that is labeled B indicates the unique
complexes formed on the site I and site III probes. All free probes
were run out of the bottom line (over night exposure gel). B,
verification of the binding specificity of the IL-6-induced
chain
complexes. Competition gel mobility shift assay was performed as
described under ``Experimental Procedures.'' Cold probes were
used at 100
excess molar as indicated in the figure. The arrow on the left side indicates the migration
position of the Stat3 complex. The arrow on the right side indicates the migration position of the IL-6-induced complexes
associated with
chain probes. SI, SII, SIII, and
2 denote the site I, site II, site
III, and
-macroglobulin gene probes, respectively. The
gel was exposed for 4 days.
The binding specificity
of the IL-6-induced complexes to chain probes were verified by
competition assays. The results shown in Fig. 4B demonstrate that the IL-6-induced complexes formed on three
chain probes could be competed away by 100
molar excess of cold
site I, site II, and site III probes (shown in Fig. 4B, lanes 5, 9, and 13, respectively) as well as
a 100
molar excess of cold
-macroglobulin probe
(shown in Fig. 4B, lanes 6, 10, and 14).
Figure 5:
The IL-6-induced chain complexes are
NEM-sensitive and contain tyrosine-phosphorylated Stat3. A,
the supershift assay was performed as described under
``Experimental Procedures.'' All nuclear protein extracts
used in the supershift assay were isolated from primary hepatocyte
treated for 5 min with IL-6. Different probes and antibodies used for
experiments are indicated as:
MG,
-macroglobulin gene probe;
-Stat3,
polyclonal rabbit anti-Stat3 antibodies (Santa Cruz); cAb,
polyclonal rabbit anti-fibrinogen antibodies as negative control
antibodies. The arrows on the left side indicate the
Stat91 and Stat3 homodimer complex migration positions and the
anti-Stat3 antibodies supershift complex migration position. The gel
was exposed for 4 days. B, the block shift assay was performed
as described under ``Experimental Procedures.'' All nuclear
protein extracts used in the gel shift assay were isolated from primary
hepatocyte treated for 5 min with IL-6. Different probes and antibodies
used for experiments are indicated as:
MG,
-macroglobulin
gene probe; 4G10, monoclonal anti-phosphotyrosine antibody
(UBI); cAb, polyclonal rabbit anti-fibrinogen antibodies as
control antibodies. The arrows on the left side indicate the dimerized Stat91 and Stat3 complex migration
positions. The gel was exposed for 4 days. C, the NEM
treatment reaction and the mock reaction were performed as described
under ``Experimental Procedures.'' All nuclear protein
extracts used in this experiment were isolated from primary hepatocyte
treated for 5 min with IL-6. Different probes and treatment used for
experiments are indicated in the figure. The arrows on the left side indicate the Stat91 and Stat3 homodimer complex
migration positions. The gel was exposed for 4
days.
The chain complexes were also
tested by anti-phosphotyrosine monoclonal antibody 4G10 (Upstate
Biotechnology, Inc.) in a gel shift assay to determine if the
phosphorylated tyrosine residue were involved in the
chain
complex formation. The results are shown in Fig. 5B. We
used the
-macroglobulin probe as the positive control (Fig. 5B, lane 1-3). The monoclonal
anti-phosphotyrosine antibody 4G10 blocked all the IL-6-induced bands
formed on the
-macroglobulin probe, which contain the
phosphorylated Stat3 or Stat91 (Fig. 5B, lane
2). Polyclonal rabbit anti-rat fibrinogen antibodies were used as
the controls and did not affect any complex formation (Fig. 5B, lane 3). Lanes 4-12 of Fig. 5B used the three
chain probes, and lanes 5, 8, and 11 show that the 4G10
antibody can also block the IL-6-induced complexes formed on the
chain site I, site II, and site III probes, respectively.
NEM
treatment and mock treatment were carried out as described above. The
results shown in Fig. 5C indicate that the NEM
treatment can remove the IL-6-induced band formed on chain
probes. Using
-macroglobulin probe as a positive
control (Fig. 5C, lanes 1-3) shows that
the IL-6-induced complexes (except Stat91 homodimer) are
NEM-sensitive(26) . Lanes 4-12 using the three
chain probes indicated that the IL-6-induced complexes were
blocked by NEM treatment (lanes 5, 8, and 11) but not by mock treatment (lanes 6, 9,
and 12). These results indicate that the IL-6-induced protein
complexes that are associated with the
chain site I, site II, and
site III probes contain the phosphorylated Stat3 component.
Both nuclear run-on studies and quantitative Northern blot
analyses have demonstrated that the fibrinogen genes are coordinately
transcribed during IL-6 stimulation(23, 27) . Because
of this strikingly coordinated regulation, one might expect the same
DNA binding complexes would be involved in their IL-6 response. This
appears not to be the case. More detailed analyses of the fibrinogen
gene promoters have shown that distinct complexes are involved in
controlling their expression. For example, the A gene has a 50-kDa
protein constitutively bound on its CTGGGA motif(19) ; however,
no such protein appears in the
gene (or B
gene). (
)Data presented here show that Stat3 is one part of the
IL-6-induced regulatory complex in the
gene. But Stat3 has not
been detected associating with the IL-6RE in the A
gene(19) . The reasons for these differences likely reside in
the flanking sequences surrounding the IL-6RE in each gene. There are
data indicating that the
and B
gene are more similar to each
other than to the A
gene. The evolutionary history of fibrinogen
provides some insight into these differences. Amino acid sequence
comparisons suggests that a primordial fibrinogen gene duplicated about
10
years ago to give a
-
chain gene and an
chain gene(28) . Additional information has indicated that the
B
and
genes then separated from one another about the time
of the appearance of the lamprey approximately 450 million years
ago(28) . In keeping with this evolutionary scheme, it appears
that the B
and
genes utilize similar regulatory proteins in
their expression, whereas the A
gene employs a different pattern
of DNA-binding proteins to up-regulate its transcription in response to
the cytokine. It is important to emphasize that even with some
individual differences in the signal pathway, all three genes are
stringently linked in their expression. How this coordination occurs
remains to be determined.
The fibrinogen gene promoter is
unique among the genes of this family in that there are three potential
IL-6RE motifs instead of a single element. Using standard reporter gene
functional analysis and mobility shift assays, we have partially
characterized the IL-6 responsive elements of this gene. The functional
assays indicate that the site II CTGGGA region confers the strongest
response to IL-6 and the site I motif is next; however, to achieve
maximal IL-6 response, all three (sites I, II, and III) CTGG(G)A
regions are required. The IL-6 response to constructs containing only a
single element was significantly lower than when at least two elements
were part of the promoter. Results of gel shift experiments provided
evidence that the three sites have similar binding affinities to the
IL-6-induced complexes. This suggests that some cooperation among the
three IL-6REs may occur during IL-6 stimulation. It is interesting that
the IL-6-induced band on site II was no more prominent than those on
sites I and III despite the results of the transfection functional
experiments indicating that site II carries the more important
functional role. A similar IL-6-induced band was also detected using
the site III probe, although it was significantly less stimulatory in
response to IL-6 in functional analysis assays.
Competition gel
shift assay using 100 excess molar cold probe of site I, site
II, and site III as well as
-macroglobulin gene probe
show that the IL-6-induced protein complexes formed on the
chain
probes are specific and exhibit characteristics similar to those of the
protein complexes that formed on the
-macroglobulin
gene probe. Supershift experiments using antibodies to Stat3 provide
evidence that the IL-6-induced complexes formed on each
chain
probe contains Stat3. This is an important finding because it links
Stat3 to another member of the class II APR protein group in addition
to
-macroglobulin. It should be emphasized that the
binding affinity of Stat3 to any of the
probes is almost 1000
times lower than that to the
-macroglobulin probe
(determined by phosphor image analysis, data not shown). Recently a
systematic analysis of the Stat-binding elements characterized the
importance of the palindrome structure TTC(N)
GAA and the
spacing between the palindrome. It was shown that the binding
specificity of different Stats to this element depends on both the
palindrome and the spacing of the half-site(29) . Analysis of
the three IL-6RE sites in the
promoter shows no strong
palindrome, but each one contains the important 5-mer spacing (CTGGG)
required for the IL-6 signal. Thus we suggest that the weakness of the
binding is due in part to a lack of a well defined palindrome motif in
these elements.
Although these findings implicate Stat3 as a part of
the IL-6-induced protein complex for up-regulating the fibrinogen
gene transcription, there still are several lines of evidence
indicating that activated Stat3 is not sufficient for regulating all
genes that contain the class II IL-6 responsive element. For example,
several different types of cytokines, including growth hormone,
epidermal growth factor, interferon
, interferon
,
platelet-derived growth factor, and IL-2, all activate
Stat3(13, 30, 31, 32) , yet none of
these cytokines exert an APR transcriptional increase in either
-macroglobulin or fibrinogen genes. Precisely how
Stat3 is involved in up-regulating gene expression is unclear. The
``strength of binding'' does not seem to be a factor because
the IL-6 response of the
-macroglobulin gene is no
greater than that of the
fibrinogen gene, yet the binding of
Stat3 to each probe is quite different. The evidence presented here
shows that the IL-6-induced transcription factor Stat3 is a participant
in regulating one of the fibrinogen genes. Undoubtedly other
DNA-binding proteins are involved and will have to be investigated
before a thorough understanding of how the IL-6 signal regulates the
fibrinogen genes.