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INTRODUCTION |
The execution of activin A biological functions depends upon
association of the cytokine with type I and type II serine/threonine kinase membrane receptors (1, 2). Activin A first binds to activin
receptor (ActR)1 type II.
This is followed by recruitment into the complex and phosphorylation of
activin receptor type IB (actRIB), which is responsible for downstream
signal transduction. Smad proteins are effectors of intracellular
signal transduction of members of the TGF
superfamily (2-5). The
mediators of activin signaling are receptor-activated Smad2 and Smad3
(6), which are present in the cytoplasm in a monomeric form. Following
activation, they undergo homodimerization and then heterodimerize with
Smad4 (Co-Smad) that lacks the C-terminal phosphorylation motif and is
therefore not activated by type I receptors (6, 7). The complex of Smad4 and receptor-activated Smads translocates to the nucleus and
binds to Smad binding elements in specific promoters (8-10). Smad3 and
Smad4 bind through their conserved MH1 domain to the palindromic
sequence GTCTAGAC (11, 12) and to CAGA-like sequences in the human
plasminogen activator inhibitor-1 (PAI-1) and
junB genes (13-15). Smads cooperate with various
transcription factors such as c-Fos and c-Jun in binding to AP-1 sites
(16). Smads also complex with Forkhead activin signal transducer
(FAST) proteins for efficient binding to specific promoters (17)
and associate with the closely related transcriptional co-activators
p300 and CBP. The latter interact with a variety of
transcription factors and thereby link these factors to the basal
transcription machinery (18-20). A hallmark in Smad signaling is
therefore their interaction with other transcription regulators.
Engagement of the activin A receptors is further followed by increased
expression of inhibitory Smad7, which binds to type I receptors and
antagonizes further signal transduction (21-23).
This general scheme of signal transduction is common to many activin
A-induced systems. One major question raised therefore is how does
activin A elicit, in different cells, distinct biological responses.
The ActR-triggered signaling cascade was found to interact with
mitogen-activated protein kinase pathways (24), and such cross-talk
among cascades may contribute to the diversity of responses. We have
shown that activin A kills tumor plasmacytomas and hybridomas (25, 26),
by competitively overcoming the survival and proliferation-promoting effects of IL-6 (27). This competition did not occur on the level of
interactions of IL-6 with its receptor complex (27), suggesting that
activin A interferes with intracellular signaling. In this hybridoma
model, cell functions, survival and proliferation, all depends on IL-6.
On the other hand, we found that activin A slows down HepG2 hepatoma
cell growth through hypophosphorylation of retinoblastoma, without
causing cell death (28). It further blocked, in these hepatoma cells,
the IL-6-induced expression of the acute phase protein haptoglobin (Hp)
(27). In the present study, we show that this happens because of
suppression of transcriptional activation of the Hp promoter by Smad proteins.
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MATERIALS AND METHODS |
Cell Lines and Cell Culture--
The human hepatoma HepG2 cell
line was maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Beth Hemeek, Israel), 20 mM L-glutamine, 60 µg/ml penicillin, 100 µg/ml streptomycin, and 50 mg/liter kanamycin.
Reagents--
Recombinant, N-terminally truncated, human IL-6
(mutein) (29) was kindly provided by Pharmacia Biocenter (Nerviano,
Italy) and was used at 100 units/ml. Recombinant activin A was obtained from the NIDDK, National Institutes of Health, National Hormone and
Pituitary Program and was used at 300 units/ml unless indicated otherwise.
Western Blotting--
The acute phase protein haptoglobin was
monitored in conditioned medium under nonreducing conditions (2% SDS,
50 mM Tris-HCl, pH 6.8, 10% glycerol, 0.01% bromphenol
blue). Extracts were subjected to 8% SDS-polyacrylamide gel
electrophoresis, blotted, and probed with specific monoclonal
antibodies (HG-36, Sigma). Chemiluminescent signals were
generated by incubation with the ECL reagent. The gels were exposed to
x-ray film.
DNA Constructs--
prHp (190)-CT plasmid, containing a 190-bp
fragment from the Hp promoter, was a gift from Dr. Heintz Baumann
(Roswell Park Cancer Institute, Buffalo, NY) (30). The 190-bp fragment
was subcloned upstream of a luciferase gene into pGL3-basic plasmid (Promega, Madison, WI). The cDNA of C/EBP
was subcloned from pBlue610, a gift from Dr. Shizuo Akira (Hyogo College of Medicine, Hyogo, Japan) into pcDNA3 expression plasmid (Invitrogen).
STAT3 was expressed in pRc/CMV (Invitrogen). Drs. Y. Zhang and R. Derynck (University of California, San Francisco) provided the
plasmids harboring Flagged Smad3, Flagged Smad4, and Flagged
Smad2 (8). The Smad genes were subcloned into pcDNA3. The PAI-1
promoter-reporter construct was a gift of Dr. D. J. Loskutoff (The
Scripps Research Institute, La Jolla, CA). Dr. Peter ten Dijke (Ludwig
Institute for Cancer Research, Uppsala, Sweden) provided Flagged Smad7
and CAactRIB (ALK4-QD) constructs. pVP22 constructs, full-length
Flagged Smad genes, were constructed in-frame, into pVP22/myc-His
Vector (Invitrogen) resulting in fusion proteins. The CMV
p300HA
construct was provided by Dr. Steven Grossman (Dana-Farber Cancer
Institute, Boston).
Luciferase Assay--
Transient transfections with the
appropriate vectors were carried out using the calcium phosphate
method. Transfection efficiency was normalized to
-galactosidase or
Renilla activities by cotransfection of 0.5 µg of CMV
-galactosidase or 2 ng of pRL-CMV expression vectors, respectively.
Cells were plated in 6-well plates (Falcon) and transfected with 0.5 µg of the reporter plasmid and different DNA constructs in a final
volume of 1.5 ml. The total amount of DNA was normalized using empty
vector. Six h after transfection, cells underwent glycerol shock and
24 h later were-serum starved and incubated in the presence or
absence of IL-6 as indicated. Cells were then harvested, and the
luciferase activity in cell lysates was determined according to
standard procedure with the aid of a Turner Designs luminometer.
Electrophorectic Mobility Shift Assay--
Cells were plated in
10-cm plates and transfected with the indicated vectors using FuGENE 6 reagent (Roche Molecular Biochemicals). Nuclear cell extracts were
prepared as described previously (31), mixed with 1-3 µg of
poly(dI-dC) (Amersham Pharmacia Biotech) in 20 µl of binding buffer
containing 110 mM KCl, 4 mM MgCl2, 4 mM Tris-HCl, pH 7.6, 4% glycerol, 0.05 mM
ZnCl2, and 0.25% bromphenol blue. The mixture was
kept on ice for 10 min, and then the end-labeled double-stranded
oligonucleotide probe was added, and incubation was continued for 20 min longer on ice. The reaction product was loaded on a 5% neutral
polyacrylamide gel and run in a Tris-glycine running buffer (25 mM Tris-HCl, pH 8.3, 190 mM glycine, and 10 mM EDTA). For supershift analysis, rabbit polyclonal
anti-C/EBP
antibodies were obtained from Santa Cruz
Biotechnology. After proteins were allowed to bind the probe,
samples were incubated with the antibodies for another 15 min at room
temperature. The oligonucleotide probe used was a double-stranded DNA
element from the haptoglobin promoter containing the C/EBP
site (top
strand) as follows: CCGACATTGTGCAAACACAGAAATGGAAG. The oligonucleotide for competition was a double-stranded non-relevant DNA element from the
haptoglobin promoter (top strand) as follows: TTTGTGGTTACTGGAACAGTCACTGACCT.
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RESULTS |
Activin A Antagonizes the IL-6-mediated secretion of the Acute
Phase Protein Hp through Inhibition of Transcription Activation of the
Hp Gene Promoter--
Activin A is a competitive antagonist of
IL-6-induced functions (27). The present study was undertaken with the
aim of elucidating the molecular basis for the ability of activin A to
block IL-6 signaling using the HepG2 hepatoma cell model. These cells
have a low basal level of secretion of APPs that is induced by
IL-6. Activin A interfered with IL-6-induced secretion of the acute phase protein, Hp, by the HepG2 hepatoma cell line (Fig.
1A). Hp is known to be
expressed in several isotypes, which accounts for the different
bands observed. Similar results were obtained when we examined the
secretion of an additional acute phase protein,
-acid glycoprotein
(27). The effect of activin A was dependent on the time of its
application relative to induction with IL-6. Thus, when added prior to
IL-6, it caused a more pronounced reduction in Hp production than
observed when activin A was added concomitantly with IL-6. By contrast,
activin A had virtually no effect when added 17 h post-IL-6
induction (Fig. 1B). The effect of activin is thus to block
Hp production rather than to increase its degradation. To test the
possibility that the effect of activin A is mediated by interference
with IL-6-induced transcription activation of the Hp gene,
we transfected HepG2 cells with a 190-bp fragment of the haptoglobin
promoter (30) upstream of a luciferase reporter and measured the
response to activin A following activation with IL-6. As shown in Fig.
2A, IL-6 triggered
transcription from the 190-bp Hp promoter fragment, whereas addition of
activin A markedly reduced this activation.

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Fig. 1.
Activin A suppresses the IL-6-induced
secretion of the acute phase protein haptoglobin from HepG2 hepatoma
cells. A, HepG2 cells were treated with IL-6 and/or
activin A (Act) under serum-free conditions. Conditioned
media were collected at 24 h, subjected to SDS-polyacrylamide gel
electrophoresis, and tested by Western blotting for the presence of
haptoglobin protein. B, HepG2 cells were treated with IL-6
and/or activin A for different time intervals. Lane 1,
medium only; lane 2, treatment with IL-6 for 24 h;
lane 3, 7-h activin A treatment prior to IL-6 (24 h);
lane 4, 4-h activin A treatment prior to IL-6 (24 h);
lane 5, 1-h activin A treatment prior to IL-6 (24 h);
lane 6, activin A and IL-6 were added together (for 24 h); lane 7, 24-h treatment with IL-6 and addition of activin
A at the last 7 h; lane 8, 24-h treatment with IL-6 and
addition of activin A at the last 4 h.
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Fig. 2.
Activin A and CAactRIB
(CAactR) both suppress transcription activation of the
Hp promoter mediated by either C/EBP or STAT3
in combination with IL-6. A luciferase reporter plasmid driven by
190 bp of the Hp promoter, pHp(190)-luc, was transfected into HepG2
cells together with CAactRIB or alone. The total amount of DNA was kept
constant by the addition of pcDNA3. 24 h after transfection,
activin A was added as indicated, and after 16 h luciferase
activity was measured. A, activin A inhibited
IL-6-induced transcriptional activation in a dose-dependent
manner. B, similar inhibition was observed when HepG2 cells
were transfected with plasmids containing cDNA encoding CAactR and
were treated with IL-6. C, Hp promoter activity induced by
C/EBP or STAT3 in combination with IL-6 was also reduced by CAactR.
D, a control experiment for the activity of the CAactR in
which the activin A-inducible PAI promoter was used. In all of the
luciferase assays, the transfection efficiency was normalized to
-galactosidase activity. Results of one experiment of three
performed are shown. Here and in the following figures, the
bars indicate the average luciferase activity ± standard
deviation of triplicate determinations.
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Constitutively active actRIB mimics activin A in inhibiting
IL-6-induced transcription and suppresses C/EBP
- and STAT3-induced transcriptional activation of the Hp promoter.
The addition of exogenous activin A to the cells could be replaced by
transfection with a constitutively active mutant form of actRIB
(CAactRIB), which almost completely abolished IL-6-induced transcription from the haptoglobin promoter (Fig. 2B). In
contrast, and as expected, CAactRIB caused marked transcriptional
activation of the control luciferase reporter construct that contain
the PAI promoter, which has a binding site for Smads (Fig.
2D). These experiments show that the signaling part of the
activin receptor complex is sufficient to execute the biological function.
Hp promoter contains DNA binding sites to several IL-6-inducible
transcription factors. It has been shown that a variety of IL-6
activities are mediated by C/EBP
and STAT proteins (32-35). Whereas
C/EBP
can be tested by direct overexpression in HepG2 cells, STAT3
must first be activated by IL-6 to enable its translocation into the
nucleus. CAactRIB reduced C/EBP
-mediated transcriptional activation
of Hp promoter and completely abolished STAT3-induced activation (Fig.
2C) indicating that activin A signaling affects IL-6-induced
transcription either on the level of these transcription factors or
downstream in the IL-6 signaling cascade. Further experiments were
aimed at identifying the molecules within the activin A signaling pathway that mediates its effect on IL-6 signal transduction. This
information could then lead to identification of the target molecule
for activin A interference within the IL-6 pathway.
Smad7 Restores IL-6 Transcriptional Activation of the Hp Promoter
Following Suppression with CAactRIB--
Signaling by actRIB recruits
the cytoplasmic Smad proteins that upon activation and phosphorylation
migrate to the nucleus and attach to specific sites in activin
A-inducible promoters. Smad7 interacts with activin A type I receptors
and blocks further downstream activation of receptor activated Smads.
It follows that if activin A operates in the suppression of IL-6
functions through Smad proteins, then Smad7 should override the effects of activin A or of CAactRIB. We found that Smad7 reverted the CAactRIB
inhibition of IL-6-induced Hp promoter transcription (Fig.
3A). Similar results were
obtained following independent activation by C/EBP
or by
IL-6-dependent STAT3-induced activation of this promoter
(Fig. 3, B and C, respectively). However, the IL-6-dependent STAT3-induced transcription was completely
restored (Fig. 3C), whereas that induced with IL-6 itself or
with C/EBP
did not return to control values. Fig. 3D
shows that PAI promoter activation by CAactRIB is completely abolished
by Smad7, verifying the ability of this protein to negatively regulate
activin A signaling.

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Fig. 3.
Smad7 relieves the transcription inhibition
of the constitutively active form of actRIB
(CAactR). The experimental details and
designations of the various additions are as described in the legend
for Fig. 2. Cells were stimulated by IL-6 (A) or,
alternatively, by transfection with STAT3 (B) or C/EBP
(C) and were co-transfected as indicated with CAactRIB
(CAactR) and/or Smad7. D, a control is
shown for the activity of Smad7 using a luciferase reporter plasmid
driven by the PAI promoter.
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We thus showed that Hp promoter activation, which is mediated by
C/EBP
or STAT3, is suppressed by activin A or by a constitutively active form of its signaling receptor. Furthermore, the transcription activation is restored by Smad7, suggesting the involvement of Smad
proteins in the suppression of IL-6-induced transcription.
Smad3 and Smad4 Suppress Transcription Activation through Hp and
-Fibrinogen Promoter Response Elements--
To directly examine the
role of Smad proteins in activin A-mediated suppression of IL-6-induced
transcription, we transfected HepG2 cells with Smad3, Smad4, or both.
Co-transfection with Smad3 and Smad4 resulted in the most pronounced
suppression of IL-6-induced Hp promoter activity (Fig.
4A). Similar experiments
performed with a combination of Smad2 and Smad4 yielded essentially
identical results (not shown). It is noteworthy that Smad3 alone also
caused significant reduction in the promoter activity, whereas Smad4 alone was inactive. These results are strongly supported by additional experiments in which Hp promoter activation was mediated by either C/EBP
(Fig. 4B) or STAT3 (Fig. 4C). The lack
of effect of Smad4 when transfected on its own could be because of its
inability to enter the nucleus. To allow for borderline activation of
the activin A pathway, we transfected the cells with CAactRIB at a concentration that does not inhibit C/EBP
-medicated transcription (25-100 ng as compared with 1.5 µg required for complete
suppression). Under these conditions Smad4 was sufficient to cause
significant suppression of C/EBP
-induced transcription from the Hp
promoter (Fig. 4D). It is noteworthy that the relationship
between the effects of Smad3 alone, as compared with Smad3 and Smad4
together, were maintained when studied on the PAI promoter, although in the case of this activin A-inducible gene, Smads caused augmentation of
transcription (Fig. 4E). We performed further
experiments with promoters from several other genes, which were either
constitutively active (CMV, GLN-LTR) or specifically
activated (IRF,
2M activated by
C/EBP
or STAT3 and IL-6). None of these promoters was suppressed in
a manner comparable with APP promoters (data not shown). Taken together
these results indicate that activin A operates by activating Smad
proteins that in turn suppress IL-6-mediated transcriptional activation
of specific promoters. Smad proteins also suppressed transcriptional
activation by the IL-6 downstream transcription activators, C/EBP
and STAT3. This phenomenon was not restricted to the Hp gene
because experiments conducted with an IL-6 response element of the
-fibrinogen promoter, which was reported to respond to C/EBP
and
STAT3 (36, 37), yielded similar results. As shown in Fig.
5, A and B,
C/EBP
- or STAT3-induced transcription from the
-fibrinogen IL-6
response element was suppressed by a combination of Smad3 and Smad4,
whereas only Smad3 on its own had a partial suppressive effect. Thus,
response elements in two independent APP gene promoters are negatively
regulated in a similar manner by Smad proteins.

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Fig. 4.
Smad proteins suppress transcription
activation of the Hp promoter. A, HepG2 cells were
co-transfected with the pHp(190)-luc reporter construct and with Smad3
and/or Smad4 expression vectors. After 24 h the cells were
harvested and stimulated with IL-6. After 12 h, luciferase
activity was measured as described in the legend for Fig. 2. These Smad
cDNA-containing constructs were also transfected into cells along
with plasmid-containing cDNA inserts encoding C/EBP
(B) or STAT3 (C). The Smad4 construct was also
transfected along with CAactRIB (D). E, a control
is shown for the activity of Smad3 and Smad4 using a luciferase
reporter plasmid driven by the activin A-inducible PAI
promoter.
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Fig. 5.
Smad proteins suppress transcription
activation of IL-6 response elements from the
-fibrinogen promoter. HepG2 cells were
co-transfected with a luciferase reporter plasmid driven by a 5XIL-6RE
from the -fibrinogen promoter and with Smads expression vectors as
indicated, along with C/EBP (A) and STAT3 (B).
Details are the same as in the Fig. 2 legend.
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To examine the effect of Smad proteins on the endogenous Hp
gene in HepG2 hepatoma cells, we used the pVP22 vector system. The VP22
protein has the ability to translocate between cells. After translation
in the cytoplasm, the expressed VP22 protein is exported from
transfected cells and translocates into the nucleus of adjacent non
transfected cells; this subsequently leads to the nuclear expression of
the transfected plasmids in a large proportion of the cells.
Full-length Flagged Smad genes were constructed in-frame into pVP22
vector as an N-terminal fusion to VP22. The cells were transfected with
these constructs and were treated with IL-6 48 h after
transfection for a period of 24 h. The end point of these
experiments was secretion of the haptoglobin protein following
induction with IL-6. Fig. 6 shows that
Smad3 together with Smad4 reduced the accumulation of haptoglobin
protein in the cultures.

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Fig. 6.
Smad proteins suppress the ability of HepG2
cells to secrete haptoglobin protein. HepG2 cells were transfected
with pVP22 plasmids containing the Smad cDNAs as indicated. After
48 h cells were harvested and treated with IL-6 for 24 h.
Conditioned media were collected, subjected to SDS-polyacrylamide gel
electrophoresis, and tested by Western blotting with anti-haptoglobin
antibodies. In addition, the amount of Smad3 was tested in Western
blotting using anti-Smad3 antibodies and was found to be identical in
the lanes representing transfection with Smad3 alone as compared with
Smad3 plus Smad4.
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The Transcription Co-activator p300 Overcomes Smad-mediated
suppression of Hp Promoter Transcription Activation--
p300 has a
histone acetyltransferase domain. By acetylation of core histones, p300
loosens the nucleosomal structure and allows access of transcription
factors to the general transcription machinery. The study of
interactions between Smads and partner proteins that form transcription
complexes revealed the functional significance of p300 as a
bridge-former that allows the execution of Smad-mediated transcriptional activation. Smad3 interacts with p300 with high affinity (18-20). p300 also binds to various transcription factors including STAT3 and C/EBP
through different segments thereby linking
them to the basal transcription machinery (38-40). Indeed, we find
that p300 by itself increased the transactivation capacity of C/EBP
and STAT3 (Fig. 7, A and
B, respectively). The formation of functional transcription
activation complexes depends upon the correct stoichiometric
concentrations of the various components. We reasoned that if Smad
competes with STAT3 and C/EBP
on binding to p300, then an excess
amount of the latter would prevent the effect of Smad. We therefore
co-transfected HepG2 cells with plasmids expressing the transcription
factors and CAactRIB along with p300. It was found that p300 reduces,
in a dose-dependent manner, the ability of CAactRIB to
interfere with IL-6 induction of transcription through either C/EBP
or STAT3 (Fig. 7, A and B, respectively). In
addition, transfection of cDNA encoding E1A, which is known to bind
p300, reduced completely the transactivation of the haptoglobin promoter by STAT3 and C/EBP
(data not shown) indicating that Smads
compete with the transcription factors for binding to p300.

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Fig. 7.
The co-activator p300 partially relieves the
inhibition of transcription from the Hp promoter mediated by CAactRIB
(CAactR). HepG2 cells were co-transfected with
pHp(190)-luc reporter plasmid and expression vectors of CAactRIB along
with C/EBP (A) and STAT3 (B). The cells were
treated with IL-6 for 12 h (in B). Luciferase activity
was measured 48 h after transfection. Details are the same as in
the Fig. 2 legend.
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Smad4 Reduces the Binding of C/EBP
to Hp Promoter DNA--
One
possible interpretation of the suppressive effect of Smad proteins on
activation of the Hp promoter is that Smads interfere, directly or
indirectly, with the binding of the relevant transcription factors to
DNA. To test this possibility, we performed EMSA assays by using
nuclear extracts obtained from cells transfected with expression
vectors. Differences between signals obtained in such EMSA assays may
result from the loading of variable amount of protein. To exclude this
possibility we verified by Western blotting that equal amounts of
C/EBP
were loaded per lane. Fig. 8
details the factors that were examined for their corresponding ability to affect the binding of C/EBP
to a 20-bp DNA oligonucleotide probe from the Hp promoter. C/EBP
bound effectively to this fragment as revealed by gel shift and supershift examination (Fig.
8A). This binding was highly specific because it was
competed out by an excess of cold promoter fragment, whereas an
irrelevant control fragment had no effect. This DNA binding was
abolished by overexpression of CAactRIB (Fig. 8B) presumably
through activation of Smads. To further examine this point, we
transfected cells with lower amount of the constitutively active
receptor to minimize the suppressive effect. Under these conditions
transfection with Smad4 leads to further inhibition of DNA binding
(Fig. 8C).

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Fig. 8.
Smad4 interferes with the binding of
C/EBP to Hp promoter DNA. A,
EMSA experiments were performed using a 33P-labeled
oligonucleotide probe containing the binding site for C/EBP from the
Hp promoter. The probe was admixed with nuclear extracts from HepG2
cells transfected with C/EBP expression vector. Supershift reaction
was performed using anti-C/EBP antibodies (Ab). A 50-fold
molar excess of the specific oligonucleotide (Oligo 50×) or
non-relevant oligonucleotide (n.r.Oligo 50×) was added as
competitor. In B and C, EMSAs of nuclear extract
of HepG2 cells transfected with the indicated expression vectors are
shown. Each section represents one representative experiment of four
performed.
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DISCUSSION |
Cytokine members of the TGF
superfamily are pleiotropic
molecules exhibiting a variety of biological functions (2). The present
study was focused on the mechanism by which activin A blocks the
secretion of the IL-6-induced APP, Hp. Our previous studies have
implied that activin A interferes with the signaling cascade induced by
IL-6 downstream to the receptor (27). We therefore focused on
the possible interactions of these intracellular transduction pathways.
It is shown here that Smad proteins are sufficient by themselves to
mediate an activin A-like suppressive effect on Hp promoter activation.
Furthermore, the suppressive effect of Smads was reproduced when the Hp
promoter was activated by overexpression with the IL-6 pathway
transcription factor, C/EBP
, alone. Smads operate therefore on the
level of C/EBP
and/or downstream in the cascade.
A constitutively active form of activin A receptor was effective
in suppressing Hp expression by the HepG2 hepatoma cell line. Whereas
activin A only partially suppressed Hp promoter activation, CAactRIB
almost abolished transcription, possibly because of the sustained
signal that a high number of constitutively active receptor molecules
per cell elicits, followed by maximal activation of downstream targets.
CAactRIB blocked transcriptional activation by C/EBP
without IL-6
stimulation, indicating that any effect of activin on IL-6 signaling
could not occur upstream to C/EBP
. We could not perform similar
experiments with STAT3 protein overexpression because this protein acts
synergistically with IL-6 to activate transcription and is insufficient
by itself. The 190-bp promoter region used in our experiments was
activated by C/EBP
and STAT3 proteins, which were very effectively
blocked by the constitutively active form of the activin type I
receptor. It therefore appears that a significant portion of the effect
of activin A is due to suppression of C/EBP
and STAT3 transcription activation.
The inhibitory Smad7 abolished the effect of CAactRIB on IL-6 or on the
transcription activators C/EBP
or STAT3. Because Smad7 has been
shown to be an inhibitor of receptor-activated Smad2 and Smad3, the
results implied the involvement of these intracellular mediators.
Direct examination by overexpression of these proteins indicated that
Smad3 on its own, and more effectively, a combination of Smad3 and
Smad4, suppressed transcriptional activation of the Hp promoter
fragment. Overexpression of Smad3 alone was sufficient to cause partial
suppression of Hp promoter, probably because of complexing with
endogenous Smad4. The use of the pVP22 vector allowed us to examine the
endogenous Hp gene. In this study a combination of Smad3 and
Smad4 transfection was needed to obtain an effect on Hp secretion.
This may be because the endogenous promoter contains sites for
binding factors that are additional to those attaching to the 190-bp
fragment, and effective suppression of these requires both Smads.
The suppressive effect of CAactRIB on C/EBP
- and STAT3-induced
transcription activation was reduced by overexpression of p300. In
other experimental systems relating to TGF
-induced functions, p300
has been shown to physically bind Smad proteins and serve as a
co-activator of transcription (18, 19). p300 might be expected to
potentiate the Smad effect rather than reduce it. However, p300
participates in transcription activation complex formation by many
other factors including C/EBP
(38) and STAT3 (39, 40). Therefore,
under conditions of limiting the amount of p300, the recruitment of
this protein by Smad3 would lead to inhibition of C/EBP
- or
STAT3-dependent transcription. By analogy to the effect of
Ski oncoprotein in attenuation of TGF
signaling, wherein this
protein reduces the recruitment of p300 into DNA binding complexes and
thus suppresses transcription, it is possible that Smad causes
dissociation of p300 from C/EBP
and STAT3 transcription activator
complexes. These data are consistent with a model in which Smad
proteins sequester p300, inhibiting C/EBP
and STAT3 functions. Activation of
-fibrinogen by IL-6 or by C/EBP
can be
similarly suppressed by p53 (41), which physically binds to p300 (42),
providing further support for the above interpretation of our data.
It is well established that Smad proteins are transcription activators
for genes inducible by TGF
superfamily members (3, 43). However,
Smad proteins are also involved in the suppression of transcription in
specific circumstances. TGIF (TG-interacting factor) and c-Ski
repress TGF
-mediated transcription by recruiting histone deacetylase
and by directly binding to Smad proteins and reducing the recruitment
of the co-activator p300 into the DNA binding complex (44-48). Thus,
in the context of TGF
-inducible genes, Smad proteins are
transcription activators but may also suppress transcription. It has
not been shown, to the best of our knowledge, that Smad proteins
mediate inhibition of transcription of promoters unrelated to the
TGF
-regulated genes. We show in this study that transcriptional
activation of response elements in the APP genes, Hp and
-fibrinogen, which is mediated by IL-6, is inhibited by
Smads. These results thus point to a novel cross-talk channel that
links the TGF
serine/threonine receptor-initiated pathway with the
IL-6 family tyrosine kinase receptor pathway. Several other studies
have implied that TGF
controls C/EBP
- or STAT3-mediated pathways.
In intestinal rat epithelial cells, TGF
caused attenuation of a
glucocorticoid-dependent increase in expression of
C/EBP
, as well as reduced binding of this transcription factor to
specific sites in the haptoglobin promoter (49) and to
-acid
glycoprotein promoter sites (50). In human T cells, TGF
antagonizes
IL-12-induced activation. Whether the effect of TGF
in this system
is mediated by blocking the phosphorylation, and hence activation of
Jak or STAT proteins, remains uncertain (51, 52).
EMSA experiments using cellular proteins showed that overexpression of
Smad4 interferes with the binding of C/EBP
to DNA. It is noteworthy
that Smad4 did not by itself interfere with transcriptional activation
of the Hp promoter (Fig. 4, A-C) unless the cells were co-transfected with CAactRIB at a concentration that does not suppress
transcription (Fig. 4D). The EMSA experiments were performed under these latter conditions. We predicted that this would assure that
the signaling machinery in the cells would be triggered and would
permit efficient translocation of Smad4 into the nucleus. This may not
occur in the transcription activation experiments in which Smad4 is
transfected into the cells on its own without the support of CAactRIB.
Thus, the particular conditions used for the DNA binding experiment
allowed us to observe a possible new function of Smad4,
i.e. a negative effect on C/EBP
binding to DNA.