Transforming Growth Factor-
and Ciliary Neurotrophic Factor
Synergistically Induce Vasoactive Intestinal Peptide Gene Expression
through the Cooperation of Smad, STAT, and AP-1 Sites*
Richard L.
Pitts,
Shuibang
Wang,
Elizabeth A.
Jones, and
Aviva J.
Symes
From the Department of Pharmacology, Uniformed Services University
of the Health Sciences, Bethesda, Maryland 20814
Received for publication, December 28, 2001, and in revised form, February 20, 2001
 |
ABSTRACT |
The cytokine ciliary neurotrophic factor (CNTF)
and transforming growth factor-
(TGF-
) both induce transcription
of the vasoactive intestinal peptide (VIP) gene through a 180-base
pair cytokine response element (CyRE) in the VIP promoter. While
CNTF induces STAT and AP-1 proteins to bind to cognate sites in the VIP
CyRE, the mechanism through which TGF-
acts to induce VIP gene
transcription is not known. Here we show that Smad3 and Smad4 proteins
can bind to two distinct sites within the VIP CyRE. These sites are
absolutely required for the induction of VIP CyRE transcription by
TGF-
. TGF-
induces endogenous Smad-containing complexes to bind
to these sites in human neuroblastoma cells. CNTF and TGF-
synergize
to induce VIP mRNA expression and transcription through the VIP
CyRE. This synergy is dependent on the Smad, STAT, and AP-1 sites,
suggesting that these two independent cytokine pathways synergize
through the cooperation of pathway-specific transcription factors
binding to distinct sites within the VIP CyRE.
 |
INTRODUCTION |
Transforming growth factor-
(TGF-
)1 and ciliary
neurotrophic factor (CNTF) have many functions in the developing and
mature nervous system. These two unrelated cytokines mediate their
effects through separate and distinct signaling cascades. CNTF, a
member of the gp130 cytokine family, utilizes a multimeric receptor
structure consisting of a GPI-linked ligand binding subunit, CNTFR-
,
and two related transmembrane signal-transducing subunits, gp130
and leukemia inhibitory factor (LIF) receptor-
(1-4). Neither of these transmembrane components have intrinsic kinase activity; instead,
they associate with the Jak/Tyk tyrosine kinases (5-7). Activation of
these kinases by ligand-induced receptor multimerization is thought to
initiate signal transduction and activation of gene expression (5, 8,
9). Cytokine stimulation induces STAT proteins to "dock" onto the
receptor, enabling their own tyrosine phosphorylation (8, 10-12).
Subsequently, STAT proteins translocate to the nucleus and bind to STAT
sites in regulated genes to provide a rapid means of activating gene
transcription (reviewed in Ref. 13). Various other signaling moieties
are also activated by CNTF including the Ras-mitogen-activated protein
kinase pathway (14-17), SHP-2 tyrosine phosphatase (18),
phosphatidylinositol 3-kinase (15, 19), and components of the AP-1
transcription factor family (20, 21).
TGF-
signals through TGF-
type I (T
R-I) and type II receptors
(T
R-II), which possess intrinsic serine-threonine kinase activity
(for reviews of TGF-
signal transduction, see Refs. 22 and 23). The
TGF-
·T
R-II complex recruits and then phosphorylates T
R-I to initiate signaling. The activated T
R-I phosphorylates the
receptor-regulated Smad proteins, Smad2 and Smad3. Phosphorylated Smads
dissociate from the receptor, complex with the co-Smad Smad4, and
translocate to the nucleus. Smads bind to specific Smad sites in
genomic regulatory regions. However, their DNA binding affinity is weak
(24), and they usually complex with other classes of transcription
factor to establish strong DNA binding to induce gene transcription of
regulated genes (25).
Despite their differences, TGF-
and CNTF share some functional
similarity. Both cytokines have neurotrophic actions, enhancing the
survival of various populations of neurons in the central and
peripheral nervous system (26-32). In addition, TGF-
enhances CNTF-mediated survival of cultured ciliary neurons, suggesting that
TGF-
may act together with CNTF in specific cell populations (33).
While these two cytokines sometimes share similar functions, the
mechanism through which they may cooperate has not been investigated. We have previously shown that CNTF and activin, a TGF-
-related cytokine, independently induce VIP gene expression through a 180-bp element in the VIP promoter termed the cytokine response element (CyRE)
(34). CNTF induces VIP gene expression through the induction of STAT
and AP-1 proteins to bind to distinct sites within the CyRE (20, 35).
These cytokine-induced proteins interact with other noninduced proteins
to bring about a robust activation of VIP transcription through
combinatorial interactions (36). While CNTF induces VIP gene expression
~50-fold, activin has a smaller effect on VIP mRNA, inducing it
~2-fold (34). However, co-treatment of CNTF and activin leads to a
synergistic activation of VIP transcription mediated through the VIP
CyRE. TGF-
also synergizes with CNTF to induce CyRE-directed
transcription (34). However, TGF-
does not induce either STAT or
AP-1 proteins to bind to the CyRE (34), and we do not know the
molecular mechanisms through which TGF-
and related cytokines induce
VIP gene expression. Understanding the TGF-
-initiated signaling
pathways that regulate the VIP gene will allow us to investigate the
mechanisms through which these two independent cytokine signals
synergize to regulate neuropeptide gene expression. In this paper, we
show that there are two Smad binding sites within the CyRE, distinct
from the AP-1 and STAT sites, that are critical to the TGF-
regulation of VIP gene expression. We further show that the Smad, STAT,
and AP-1 sites all contribute to the synergistic interaction between
CNTF and TGF-
in the induction of VIP gene expression.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Cell culture reagents were obtained from
Mediatech (Herndon, VA), fetal bovine/horse serum from Life
Technologies, Inc., and culture plates from Costar (Corning, NY).
Recombinant human CNTF was a gift from Regeneron Pharmaceuticals
(Tarrytown, NY), and TGF-
was purchased from R & D Systems
(Minneapolis, MN). Oligonucleotides were synthesized on a PE Applied
Biosystems 394 synthesizer by the Uniformed Services University of the
Health Sciences in-house oligonucleotide facility. Anti-Smad2/3
antiserum was obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). The Gal4-Smad fusion plasmids, pG5-E1Bluc, and bacterial
expression vectors for GST-Smad4 and GST-Smad3
C were obtained from
Dr. R. Lechleider (Department of Pharmacology, Uniformed Services
University of the Health Sciences) (37).
Plasmids--
Details of Cy1luc, the Cyluc deletion series,
3×G3 luc, and 3×G2 luc have all been described (35). The series of
3-bp substitution mutants of Cy1luc was amplified from Cy1luc as
described previously (36). The plasmid Cy1mS5 is identical to Cy1
mg11luc. Cy1 mg17luc was constructed by polymerase chain reaction
site-directed mutagenesis (38) with the oligonucleotides 5'-CAAC
TGGGAAACAAATTTCCATCGATTTTGAAACTTAATTC-3' and
5'-GAATTAAGTTTCAAAATCGATGGAAATTTGTTTCCCAGTTG-3'. These
oligonucleotides were paired, with either A1 or A4 (35), and
Cy1luc as template to create new fragments. The fragments were
gel-purified and used as template in a subsequent polymerase chain
reaction with oligonucleotides A1 and A4 as primers to create
Cy1mg17luc. This plasmid, Cy1mg17luc, was used as template with primers
A1 and mS5 (36) to create Cy1mg17mg11. Cy1mg17mg11luc was then used as
template DNA to construct the further mutated Cy1mG17mG11mG3luc,
Cy1mG17mG11mG2luc, and Cy1mG17mG11mG3mG2luc using the
CLONTECH Transformer site-directed mutagenesis kit
(CLONTECH Inc. Palo Alto, CA). All plasmids were sequenced to confirm their identity. SBE-luc contained four copies of
the Smad-binding element (SBE) consensus (39). The Gal4-Smad fusion
plasmids, pG5-E1Bluc, and bacterial expression vectors for GST-Smad4
and GST-Smad3
C have been described previously (37).
Cell Culture and Transfection--
NBFL cells were maintained
and transfected as described previously (40). Cells were plated at
1.5 × 105 cells/well in six-well plates and
transfected overnight by calcium phosphate precipitation. Each well
received 1 µg of luciferase reporter construct, 0.5 µg of
EF-
-galactosidase, and 2.5 µg of carrier DNA. Cytokines were added
in serum-free medium, 6 h after the DNA precipitate was
removed, for 40 h before cell harvesting. Samples were assayed for
luciferase activity (41) and
-galactosidase activity (Galacto-Light
Plus kit, Tropix Inc, MA). Luciferase activity was normalized to
-galactosidase activity to control for transfection efficiency.
RNA Isolation and Analysis--
Total cytoplasmic RNA was
isolated from NBFL cells after lysis with Nonidet P-40 and transferred
to nylon membranes as previously described (42). Northern blots were
hybridized with a 580-bp HindIII/EcoRI fragment
of human VIP cDNA (43) and rehybridized with a probe for the
unregulated internal reference gene cyclophilin (44). Blots were
digitized using a Storm PhosphorImager, with ImageQuant software
(Molecular Dynamics, Inc., Sunnyvale, CA). The relative densitometric
readings were normalized to cyclophilin mRNA to account for loading
differences between lanes.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSAs were
performed as previously described (35). NBFL cells were grown to
confluency and serum-starved overnight before treatment with TGF-
for the times indicated. GST-Smad fusion proteins were prepared as
described (37). Nuclear extracts were prepared, and binding reactions
performed as previously described (35). Synthetic complementary
oligonucleotides with GGG overhangs were annealed and labeled with
[
-32P]dCTP using Moloney murine leukemia virus reverse
transcriptase (Promega, WI). When used, competitor oligonucleotides or
antibodies were incubated with the nuclear extracts for 10 min at room
temperature prior to the addition of probe.
 |
RESULTS |
We have previously shown that TGF-
induction of VIP gene
transcription is mediated through the VIP CyRE. To determine which regions within the 180-bp CyRE are important for the TGF-
-mediated transcriptional induction, we transfected NBFL neuroblastoma cells with
a series of CyRE deletion constructs. These plasmids contain various
regions of the CyRE upstream of a basal RSV promoter driving expression
of the luciferase reporter gene (35). As described previously,
luciferase activity in cells transfected with the full-length Cy1luc
was induced after treatment with TGF-
~4-fold (34). Deletion of 28 bp at the 3'-end of the CyRE either from the full-length construct or
from two other plasmids with deleted 5'-ends reduced the
transcriptional induction of CyRE luciferase reporter plasmids by over
60% (Fig. 1). However, deletion of up to
50 bp from the 5'-end of the CyRE did not significantly reduce the
induction of luciferase activity driven by these plasmids in response
to TGF-
. These data suggest that a region within the most 3' 28 bp
of the CyRE is important to the TGF-
-mediated induction of VIP CyRE
transcription.

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Fig. 1.
Deletion of the 3' end reduces the
TGF- inducibility of the CyRE. NBFL
cells, transfected with the luciferase reporter plasmids shown, were
either left untreated or treated for 40 h with TGF- (2.5 ng/ml)
before harvesting and analysis of luciferase and -galactosidase
activity. Data are presented as -fold induction of luciferase activity
normalized to -galactosidase activity (mean ± S.E.) of three
independent experiments.
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|
To examine more precisely the sequences within this 3' region that
contribute to the TGF-
induction of VIP transcription, we
transfected NBFL cells with CyRE-luciferase plasmids containing a
sequential series of 3-bp mutations of the most 3' 30 bp (Fig. 2). TGF-
was unable to induce
transcriptional activity in cells transfected with either Cy1mS4luc or
Cy1mS5luc (Fig. 2). These plasmids contain sequential mutations of the
sequence GTCTGA, which, read inverted on the minus strand, contains a
CAGA box (TCAGAC). In other promoters, this CAGA motif can bind Smad
proteins and mediate TGF-
induced transcription (39, 45). The
mutations present in Cy1mS7luc and Cy1mS8luc reduced but did not
eliminate TGF-
-induced transcription (Fig. 2), suggesting that these
mutated sequences may also contribute to the TGF-
induction of VIP
CyRE transcription. Mutations in other regions within the 3' CyRE
resulted in reduced unstimulated and TGF-
-stimulated luciferase
activity but no significant alteration in the overall TGF-
induction
(Fig. 2). The exception to this, the large TGF-
induction of
Cy1mS6luc, was not reproducible. These data suggest that the sequence
TCAGAC is critical to the ability of TGF-
to induce transcription
through the VIP CyRE.

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Fig. 2.
Mutation of specific sequences within the
3'-end reduce the TGF- inducibility of the
CyRE. NBFL cells, transfected with the luciferase reporter
plasmids shown, were either left untreated or treated for 40 h
with TGF- (2.5 ng/ml) before harvesting and analysis of luciferase
and -galactosidase activity. Data are of a representative experiment
performed in triplicate. Data are presented as normalized luciferase
activity (mean ± S.E.) and as -fold induction in luciferase
activity. The experiment was repeated three times with similar
results.
|
|
We then examined the rest of the CyRE sequence to look for other
regions that might have similarity to the CAGA site at the 3'-end of
the CyRE. Interestingly, we found one other region, toward the center
of the CyRE, that also contained the core CAGA sequence. This sequence
(TCCAGACAT) is located
1250 bp upstream of the transcription start
site. To determine whether Smad proteins could bind to these CAGA
containing sequences from the VIP CyRE, we incubated purified GST-Smad
fusion with probes from these regions, P17 and P11 (Table I). EMSA
analysis indicated that GST-Smad3
C bound to the CAGA-containing
probes, P17 and P11, and to a control SBE
but not to an adjacent,
non-CAGA-containing probe, P18 (Fig. 3C). Binding of GST-Smad3
C
to either P17 or P11 was specifically competed by a 100-fold
molar excess of nonlabeled wild type oligonucleotide but not by the
same amount of these sequences with 3-bp mutations in their CAGA boxes
(Fig. 3D). GST-Smad4 bound very weakly to either P17 or P11,
despite strong binding to the control SBE (Fig. 3E).
Truncated GST-Smad4
C, without the C-terminal MH2 domain, did not
bind more strongly than full-length GST-Smad4 (data not shown),
suggesting that it was not the presence of the MH2 domain that
inhibited Smad4 binding to the CyRE sequences. Taken together, these
data show that two sites within the VIP CyRE are able strongly and
specifically to bind Smad3 but that these same sites bind Smad4 only
weakly.

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Fig. 3.
Smad proteins can bind to two distinct sites
with in the VIP CyRE. A, schematic representation of
the CyRE, showing the locations of known and putative transcription
factor binding sites, together with the positions of EMSA probes.
B, alignment of sequence of probes used in EMSA.
C, EMSA with purified GST (G) or GST-Smad3 C
(S3) with three different probes from the CyRE and the
positive control SBE. D, EMSA with GST-Smad3 C binding to
two probes from the CyRE. Competing oligonucleotides were present at a
100-fold molar excess. E, EMSA with purified GST-Smad fusion
proteins binding to SBE, P17, and P11 probes. The arrow
indicates the position of weak GST-Smad4 binding to P17 and P11.
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To assess the role of these two CyRE Smad binding sites in mediating
TGF-
inducibility of CyRE-directed transcription, we examined the
ability of TGF-
to induce transcription of luciferase reporter
plasmids containing mutations in either or both of these Smad sites.
Identical mutations to those that eliminated the ability of Smad
proteins to bind to P17 or P11 were introduced into the wild type
Cy1luc plasmid. Transfection of these plasmids into NBFL cells
indicated that mutation of either P17 or P11 attenuated the ability of
TGF-
to induce CyRE-mediated transcription (Fig. 4). Mutation of both P17 and P11
completely eliminated the ability of TGF-
to induce CyRE-driven
transcription. Thus, while each CyRE Smad binding site contributes,
both sites are necessary to TGF-
induction of CyRE-driven
transcription.

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Fig. 4.
Mutation of the CyRE Smad sites eliminates
the TGF- inducibility of the CyRE. NBFL
cells, transfected with the luciferase reporter plasmids shown, were
either left untreated or treated for 40 h with TGF- (2.5 ng/ml)
before harvesting and analysis of luciferase and -galactosidase
activity. Data are of a representative experiment performed in
triplicate (mean ± S.E.). The experiment was repeated three times
with similar results.
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To determine whether TGF-
induces endogenous proteins within NBFL
cells to bind to these Smad sites, we prepared nuclear extract from
untreated and TGF-
-treated NBFL cells. After 1 h of treatment,
TGF-
strongly induced NBFL nuclear protein binding to probes
containing the CyRE Smad binding sequences (Fig.
5). The probe, P21, is a truncated
version of P17. The TGF-
-induced p21-binding complex is competed
specifically by a 100-fold molar excess of unlabeled probe and also by
a similar molar excess of P17 and P11. It is not competed by
oligonucleotides with mutations in their CAGA boxes that were unable to
bind GST-Smad proteins (Fig. 5A). These data suggest that in
NBFL cells, TGF-
activates Smad proteins to translocate to the
nucleus and bind to Smad binding sites within the VIP CyRE.
Confirmation of the composition of these TGF-
-induced binding
complexes was obtained by demonstrating that an antibody recognizing
Smad2 and Smad3 was able to interfere with binding of these complexes
to the P21 and P11 probes (Fig. 5). Thus, taken together, our data show
that TGF-
induces Smad proteins to bind to two sites with the VIP
CyRE and that this binding is critical for TGF-
induction of VIP
transcription.

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Fig. 5.
TGF- induces a Smad
containing complex to bind to a site in the VIP CyRE. EMSAs with
nuclear extract prepared from NBFL cells either untreated or treated
with TGF- (5 ng/ml) for 1 h. Competing oligonucleotides were
present at a 100-fold molar excess. 2 µg of the antiserum that
recognizes Smad2 and Smad3 (Upstate Biotechnology, Inc.) was added to
the nuclear extract 10 min before the addition of probe.
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We first demonstrated the VIP CyRE to be a response element for the
gp130 cytokines (in particular for CNTF) (35) and subsequently that
CNTF synergized with activin to induce VIP mRNA (34). Since activin
and TGF-
utilize very similar pathways to regulate gene expression,
we wanted to confirm that CNTF would also synergize with TGF-
to
induce VIP mRNA. Analysis of VIP mRNA expression in NBFL cells
by Northern blotting showed that TGF-
treatment alone induced VIP
mRNA in a dose-dependent manner. 1 ng/ml TGF-
induced VIP mRNA 2.8-fold, and 10 ng/ml led to a 12.4-fold
induction of VIP mRNA (Fig. 6). As we
had previously observed with activin, the TGF-
induction is
significantly less robust than that elicited by CNTF, which produced a
55-fold induction in VIP mRNA. When NBFL cells were treated with
CNTF together with TGF-
, VIP mRNA was markedly induced: 147-fold
by CNTF with 1 ng/ml TGF-
and 257-fold by CNTF together with 10 ng/ml TGF-
(Fig. 6). Thus, TGF-
acts synergistically with CNTF to
induce VIP mRNA.

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Fig. 6.
TGF- synergizes with
CNTF to induce VIP mRNA expression. A, Northern
blot of cytoplasmic RNA (20 µg) isolated from NBFL cells treated with
TGF- and/or CNTF (25 ng/ml) for 48 h. B, graphical
representation of data from Northern blot shown in A,
quantitated on a PhosphorImager. VIP mRNA levels are normalized to
those of cyclophilin mRNA and presented as a relative ratio.
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TGF-
and CNTF both induce VIP gene transcription through inducing
proteins to bind to specific sequences within the VIP CyRE, yet they
activate different transcription factors. We wanted to assess the
contribution of sites important to either the CNTF or TGF-
pathways
to the synergistic signaling of these two independent cytokines. We
therefore introduced additional mutations at the STAT and/or AP-1 sites
into the Cy1luc reporter with both Smad sites mutated and compared the
activity of all of these mutated luciferase reporters to the wild type
Cy1luc plasmid. NBFL cells transfected with Cy1luc reproduced the
synergistic effect of CNTF and TGF-
on VIP mRNA, mediating
strong inducibility by CNTF, less by TGF-
, and very marked
synergistic signaling by cotreatment with the two cytokines (Fig.
7). In cells transfected with
Cy1mg17mg11 (containing mutations of both CyRE Smad sites),
TGF-
was no longer able to induce luciferase activity, as shown
previously (Fig. 4). Interestingly, the CNTF-induced transcription
driven by Cy1mg17mg11 was reduced by 60% in comparison with Cy1luc,
and there was no significant difference in transcriptional induction
after cotreatment with both cytokines from that of CNTF alone (Fig. 7).
Transcriptional activity driven by a luciferase reporter with mutations
in both Smad sites and the AP-1 site (Cy1mg17mg11mg2) was not induced by TGF-
but still retained CNTF induction. The synergy between TGF-
and CNTF was no longer evident. Plasmids containing
mutations of the Smad and STAT sites (Cy1mg17mg11mg3) or of the Smad,
STAT, and AP-1 sites (Cy1mg17mg11mg3mg2) did not respond to stimulation by either cytokine alone or together. These data suggest that CNTF and
TGF-
synergistically induce CyRE transcription through stimulating a
combination of Smad, STAT, and AP-1 proteins.

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Fig. 7.
Synergistic signaling of
TGF- and CNTF requires the Smad, STAT, and
AP-1 sites. NBFL cells, transfected with the luciferase reporter
plasmids shown, were either left untreated or treated for 40 h
with TGF- (2.5 ng/ml) and/or CNTF (25 ng/ml) before harvesting and
analysis of luciferase and -galactosidase activity. Data are
presented as -fold induction of luciferase activity normalized to
-galactosidase activity (mean ± S.E.) of three independent
experiments.
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|
CNTF and TGF-
stimulate very different signaling pathways to
activate gene expression. However, the possibility of cross-talk between these pathways exists at many different levels. Smad proteins, while initially phosphorylated by the receptor, may also be
phosphorylated by cytoplasmic kinases, such as the extracellular
signal-regulated kinases (37, 46). To investigate whether CNTF
signaling may contribute to Smad activation, independent of any
potential Smad-DNA binding effects, we utilized Gal4-Smad fusion
proteins. Expression vectors for Gal4-Smad fusion proteins were
co-transfected with a reporter containing multimerized Gal4 DNA binding
sites, pG5-E1B-luc. As previously published (37, 47), TGF-
induced
transcriptional activation mediated by Gal4-Smad2, Gal4-Smad3, and
Gal4-Smad4 but not by Gal4 alone (Fig.
8A). TGF-
induction of
Gal4-Smad3 transcriptional activity was the most robust. CNTF did not
induce transcription by any Gal4-Smad fusion proteins. However, CNTF and TGF-
co-treatment significantly induced Gal4-Smad3-mediated transcription over that of TGF-
alone. While there was a trend toward this difference with Gal4-Smad2 and Gal4-Smad4, this trend was
not significant. These data suggest that while CNTF signals alone do
not induce Smad transcriptional activation, CNTF may enhance TGF-
's
activation of Smad3. However, CNTF and TGF-
co-treatment did not
enhance TGF-
's induction of the multimerized SBE luciferase reporter, SBE luc (Fig. 8D). These data suggest that CNTF
does not significantly alter the TGF-
activation of endogenous Smad proteins. Thus, the effects of CNTF signals on TGF-
induction of
Smad transcriptional activity are minimal.

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Fig. 8.
CNTF and TGF- signal
through independent pathways. NBFL cells were transfected as shown
and were left untreated or treated for 40 h with TGF- (2.5 ng/ml) and/or CNTF (25 ng/ml) before harvesting and analysis of
luciferase and -galactosidase activity. A, NBFL cells
were transfected with expression plasmids for the Gal4-Smad fusion
plasmids together with the Gal4 reporter plasmid, pG5-E1B-luc,
containing five copies of the Gal4 DNA binding site. Data are from a
representative experiment performed in triplicate (mean ± S.E.)
The experiment was repeated four times with similar results. Data were
analyzed by one way analysis of variance and evaluated using the
Bonferroni multiple comparisons test. Significant differences between
normalized luciferase activity in lysates from untreated cells and
those in lysates from cytokine-treated cells within each group are
indicated: *, p < 0.05; **, p < 0.01;
***, p < 0.001. Additionally, there is a significant
difference (p < 0.001) in the normalized luciferase
activity from Gal4-Smad3-transfected cells, between those treated with
TGF- and those with CNTF plus TGF- . B, C,
and D, NBFL cells were transfected with the luciferase
reporter plasmids shown. Experiments were repeated a minimum of twice
with similar results.
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|
To determine whether TGF-
may affect CNTF activated pathways, we
examined the effects of cytokine cotreatment on luciferase reporters
driven by multimerized STAT (G3) or AP-1 (G2) sites. We have previously
shown that CNTF does not induce transcription driven by multimerized
AP-1 sites and only minimally induces transcription driven by a
multimerized VIP-STAT site, 3×G3 luc (here 2-fold; Fig. 8C)
(20, 35). TGF-
treatment did not induce transcription driven by
either multimerized STAT or AP-1 sites and in fact inhibited the basal
levels of transcription of these plasmids. This effect was partially
due to a slight stimulation by TGF-
of the co-transfected normalization plasmid, EF-
-galactosidase (data not shown). TGF-
did not alter the CNTF induction of STAT-mediated transcription, nor did it induce transcription driven by the multimerized AP-1 site
(3×G2-luc). Thus, TGF-
signals do not alter the CNTF-stimulated pathways we have examined. Our data suggest that CNTF and TGF-
independently activate distinct signal pathways, leading to the stimulation of specific transcription factors that bind to consensus sites on the VIP CyRE.
 |
DISCUSSION |
The VIP CyRE is able to mediate independent and synergistic
signaling by the gp130 and TGF-
family of cytokines. This 180-bp sequence is able to respond to these two distinct classes of cytokines because it contains within it consensus sequences for transcription factors that are rapidly activated by cytokine signaling. Hence, CNTF
induces STAT and AP-1 proteins to bind to their specific sites within
the CyRE, and TGF-
induces Smad proteins to bind to Smad sites also
contained in the CyRE. The information encoded within the VIP CyRE
therefore allows signaling by these cytokine classes to interact by the
cooperation of transcription factors binding to DNA within a relatively
small region.
We have shown that a C-terminal truncated Smad3 protein can bind to two
distinct sites within the VIP CyRE (Fig. 3). Mutation of both of these
sites abolishes the ability of TGF-
to induce transcription through
the VIP CyRE (Fig. 4). Both Smad sequences have strong homology to Smad
binding sites in many other genes including the PAI
and junB promoters (45, 48). From EMSA studies it appears
that the sequence within P17, CCAGACA, has higher affinity for
GST-Smad3
C than the sequence within P11, TCAGACT (data not shown).
Additionally, we have shown that in NBFL cells TGF-
induces nuclear
protein complexes to bind to these two Smad sites within the CyRE.
These complexes are removed by antiserum recognizing both Smad2 and
Smad3 (Fig. 5). Smad2 does not bind to DNA (49), so these binding
complexes probably contain Smad3. However, we cannot rule out the
involvement of Smad2 in transcriptional activation of the VIP gene by
TGF-
. GST-Smad4 can bind weakly to the two CyRE Smad sites. Although
Smad4 can bind to sites with a CAGA box (39), it also has shown
preference for a GC-rich site, similar to that to which the
Drosophila Smad homolog Mad binds in the promoter of the
vestigial gene (24). Our results indicate a discrepancy between the
affinity of Smad3 and Smad4 for the VIP CyRE Smad sites, suggesting
that Smad3 and Smad4 proteins have different sequence specificity even
within binding to CAGA box sites. Removing the C-terminal MH2 domain
from Smad4 did not increase binding to the CyRE Smad sites, in contrast
to the results of Jonk et al. (48), who show much improved
binding of GST-Smad4
C over full-length GST-Smad4 to sites in the
JunB promoter. Thus, Smad4 may require other proteins to assist its
binding to the VIP CyRE.
Smad proteins bind DNA with low affinity (49). Therefore, to achieve
high affinity interaction with specific DNA sequences, Smad proteins
usually form complexes with other transcriptional co-factors or bind as
multimeric proteins to repeats of the CAGA motif (50, 51). Cooperative
binding of Smad proteins confers a greater level of specificity,
conferring dependence on the specific promoter sequence of each gene
and the availability of co-factors with which to bind. The distance
between the two VIP CyRE Smad sites (85 bp) suggests that Smad proteins
require other proteins to achieve high affinity binding. Thus, Smad3,
possibly together with Smad2 and Smad4, may bind to each site in
complex with an as yet unknown transcriptional co-factor. Indeed, in
EMSA experiments, we have seen a larger TGF-
-induced nuclear protein
complex binding to the longer P17 probe than binds to P21 (data not
shown). However, our data also show that deleting or mutating only one
Smad site significantly reduces the ability of TGF-
to induce
transcription through the VIP CyRE (Figs. 1 and 4). Thus, one Smad site
alone together with its adjacent sequence is not sufficient to confer full TGF-
induction of VIP transcription. The two Smad sites may
functionally cooperate through the looping out of intervening sequences
to form a greater transcriptional activating complex with which to
recruit co-activators.
While the Smad sites within the VIP CyRE are critical for
TGF-
-mediated induction of CyRE transcriptional activity, the AP-1 site is an additional site through which TGF-
may act. We have previously shown that mutation of the AP-1 site in the VIP CyRE reduces
activin-mediated induction of CyRE-directed transcription ~50% (34).
However, our observation that mutation of the CyRE Smad sites
eliminated TGF-
stimulation of CyRE transcription suggests that the
Smad sites are more critical to the CyRE response to TGF-
than the
AP-1 site. Our data are in contrast to studies on the collagenase
promoter, where mutations in the AP-1 site within the collagenase I
promoter reduce TGF-
-mediated induction of this gene to a much
greater extent than mutations in any or all of the Smad sites (52).
Thus, the relative contributions of the AP-1 and Smad sites to
transcriptional induction by TGF-
appear to be gene-specific.
One possible mechanism mediating the synergy between CNTF and TGF-
is the convergence of their signaling pathways to activate specific
transcription factors. Our data suggest that some kinase activation by
CNTF may contribute to Smad transcriptional activation when already
activated by TGF-
(Fig. 8A). However, this effect is
likely to be minimal due to the lack of synergistic signaling by CNTF
and TGF-
in the activation of a transcriptional reporter composed of
multimerized Smad sites (Fig. 8D). Thus, synergy between CNTF and TGF-
is not mediated solely through Smad proteins; nor can
TGF-
synergize with CNTF in activation of a multimerized STAT
reporter. Thus, the synergy between CNTF and TGF-
is not mediated by
the action of one cytokine signaling directly to the transcription
factor activated by the second cytokine.
CNTF and TGF-
activate pathway-specific transcription factors that
translocate to the nucleus to activate gene transcription through the
VIP CyRE. As STAT, Smad, and AP-1 sites contribute to the synergistic
signaling by these independent cytokines, it seems possible that these
activated transcription factors are able to form a more stable
transcriptional activation complex when activated together than when
either pathway alone is activated. Such a transcriptional activation
complex would provide a base for interaction with co-activators such as
CBP/p300. As Smad, AP-1, and STAT proteins all interact directly with
CBP (53-56), CBP may act as a bridging molecule to mediate the synergy
between these pathways. Indeed, CBP/p300 is implicated in the
synergistic interaction between BMP2 and LIF induction of the GFAP
promotor in fetal neuroepithelial cells through bridging LIF-induced
STAT3 with the BMP2-induced Smad1 (57). Thus, we hypothesize that transcription factors activated by both cytokine signaling pathways act
together with constitutive and cell-specific transcription factors to
form a more stable surface with which to recruit co-activators.
CNTF and TGF-
together with their related cytokines have important
roles in neuronal survival, development, and mediation of some of the
responses of the nervous system to injury (32, 58). Neuronal and
nonneuronal cells within the nervous system can respond to cytokines of
both families dependent on the specific receptors expressed. Each
cytokine acts in cooperation with its environment. Thus, it is
important to understand the molecular details through which specific
cytokines interact to influence cellular functions. Gp130 cytokines may
interact with certain members of the TGF-
cytokine family in the
glial reaction to injury, in astrocyte differentiation, and in neuronal
survival. Indeed, BMP2 and LIF synergize in the development of
astrocytes (57) from neuroepithelial cells, and CNTF and TGF-
cooperate to mediate the survival of chick ciliary ganglion neurons
(33). The regulation of VIP gene expression is another example of
functional cooperation between these two cytokine families. TGF-
is
generally considered a modulator cytokine, having different effects
dependent on the cellular context. Thus, its ability to synergize with
CNTF allows target cells enhanced ability to regulate their responses to low doses of cytokines. Such combinatorial action increases the
repertoire of options available to the cell and makes them more finely
tuned to the environmental cytokine milieu.
 |
ACKNOWLEDGEMENTS |
We thank Fern Murdoch and Bob Lechleider and
members of their laboratories for many helpful discussions and
suggestions. We also gratefully acknowledge assistance and suggestions
from Liliana Attisiano. We thank Regeneron Pharmaceuticals for
supplying the CNTF.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R29 NS-35839 and Uniformed Services University of the Health Sciences intramural support (to A. J. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Dept. of Pharmacology,
Uniformed Services University of the Health Sciences, 4301 Jones Bridge
Rd., Bethesda, MD 20814. Tel.: 301-295-3234; Fax: 301-295-3220; E-mail:
Asymes@usuhs.mil.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M011759200
 |
ABBREVIATIONS |
The abbreviations used are:
TGF, transforming
growth factor;
CNTF, ciliary neurotrophic factor;
LIF, leukemia
inhibitory factor;
T
R-I and -II, TGF-
type I and II receptor,
respectively;
CyRE, cytokine response element;
STAT, signal transducers
and activators of transcription;
bp, base pair(s);
EMSA, electrophoretic mobility shift assay;
GST, glutathione
S-transferase;
VIP, vasoactive intestinal peptide;
SBE, Smad-binding element.
 |
REFERENCES |
1.
|
Ip, N. Y.,
Nye, S. H.,
Boulton, T. G.,
Davis, S.,
Taga, T.,
Li, Y.,
Birren, S. J.,
Yasukawa, K.,
Kishimoto, T.,
Anderson, D. J.,
Stahl, N.,
and Yancopoulos, G. D.
(1992)
Cell
69,
1121-1132[Medline]
[Order article via Infotrieve]
|
2.
|
Davis, S.,
Aldrich, T. H.,
Stahl, N.,
Pan, L.,
Taga, T.,
Kishimoto, T.,
Ip, N. Y.,
and Yancopoulos, G. D.
(1993)
Science
260,
1805-1808[Medline]
[Order article via Infotrieve]
|
3.
|
De Serio, A.,
Graziani, R.,
Laufer, R.,
Ciliberto, G.,
and Paonessa, G.
(1995)
J. Mol. Biol.
254,
795-800[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Carpenter, L. R.,
Yancopoulos, G. D.,
and Stahl, N.
(1998)
Adv. Protein Chem.
52,
109-140[Medline]
[Order article via Infotrieve]
|
5.
| Guschin, D., Rogers, N., Briscoe, J., Witthun, B., Watling, D., Horn,
F., Pellegrini, S., Yasukawa, K., Heinrich, P., Stark, G., Ihle,
J., and Kerr, I. (1995) EMBO J. 1421-1429
|
6.
|
Narazaki, M.,
Witthuhn, B. A.,
Yoshida, K.,
Silvennoinen, O.,
Yasukawa, K.,
Ihle, J. N.,
Kishimoto, T.,
and Taga, T.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
2285-2289[Abstract]
|
7.
|
Stahl, N.,
Boulton, T. G.,
Farruggella, T.,
Ip, N. Y.,
Davis, S.,
Witthuhn, B. A.,
Quelle, F. W.,
Silvennoinen, O.,
Barbieri, G.,
Pellegrini, S.,
Inle, J. N.,
and Yancopoulos, G. D.
(1994)
Science
263,
92-95[Medline]
[Order article via Infotrieve]
|
8.
|
Stahl, N.,
Farruggella, T. J.,
Boulton, T.,
Zhong, Z.,
Darnell, J.,
and Yancopoulos, G. D.
(1995)
Science
267,
1349-1353[Medline]
[Order article via Infotrieve]
|
9.
|
Stahl, N.,
and Yancopoulos, G. D.
(1994)
J. Neurobiol.
25,
1454-1466[Medline]
[Order article via Infotrieve]
|
10.
|
Bonni, A.,
Frank, D. A.,
Schindler, C.,
and Greenberg, M. E.
(1993)
Science
262,
1575-1579[Medline]
[Order article via Infotrieve]
|
11.
|
Lutticken, C.,
Wegenka, U. M.,
Yuan, J.,
Buschmann, J.,
Schindler, C.,
Ziemiecki, A.,
Harpur, A. G.,
Wilks, A. F.,
Yasukawa, K.,
Taga, T.,
Kishimoto, T.,
Barbieri, G.,
Pellegrini, S.,
Sendtner, M.,
Heinrich, P. C.,
and Horn, F.
(1994)
Science
263,
89-92[Medline]
[Order article via Infotrieve]
|
12.
|
Zhong, Z.,
Wen, Z.,
and Darnell, J. E. J.
(1994)
Science
264,
95-98[Medline]
[Order article via Infotrieve]
|
13.
|
Heinrich, P. C.,
Behrmann, I.,
Muller-Newen, G.,
Schaper, F.,
and Graeve, L.
(1998)
Biochem. J.
334,
297-314[Medline]
[Order article via Infotrieve]
|
14.
|
Schwarzschild, M. A.,
Dauer, W. T.,
Lewis, S. E.,
Hamill, L. K.,
Fink, J. S.,
and Hyman, S. E.
(1994)
J. Neurochem.
63,
1246-1254[Medline]
[Order article via Infotrieve]
|
15.
|
Boulton, T. G.,
Stahl, N.,
and Yancopoulos, G. D.
(1994)
J. Biol. Chem.
269,
11648-11655[Abstract/Free Full Text]
|
16.
|
Schiemann, W. P.,
and Nathanson, N. M.
(1994)
J. Biol. Chem.
269,
6376-6382[Abstract/Free Full Text]
|
17.
|
Giordano, V.,
De Falco, G.,
Chiari, R.,
Quinto, I.,
Pelicci, P. G.,
Bartholomew, L.,
Delmastro, P.,
Gadina, M.,
and Scala, G.
(1997)
J. Immunol.
158,
4097-4103[Abstract]
|
18.
|
Schiemann, W. P.,
Bartoe, J. L.,
and Nathanson, N. M.
(1997)
J. Biol. Chem.
272,
16631-16636[Abstract/Free Full Text]
|
19.
|
Chen, R. H.,
Chang, M. C.,
Su, Y. H.,
Tsai, Y. T.,
and Kuo, M. L.
(1999)
J. Biol. Chem.
274,
23013-23019[Abstract/Free Full Text]
|
20.
|
Symes, A. J.,
Gearan, T.,
Eby, J.,
and Fink, J. S.
(1997)
J. Biol. Chem.
272,
9648-9654[Abstract/Free Full Text]
|
21.
|
Wang, Y.,
and Fuller, G. M.
(1995)
Gene (Amst.)
162,
285-289[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Massague, J.,
and Wotton, D.
(2000)
EMBO J.
19,
1745-1754[Abstract/Free Full Text]
|
23.
|
Miyazono, K.,
ten Duke, P.,
and Heldin, C. H.
(2000)
Adv. Immunol.
75,
115-157[Medline]
[Order article via Infotrieve]
|
24.
|
Kim, J.,
Johnson, K.,
Chen, H. J.,
Carroll, S.,
and Laughon, A.
(1997)
Nature
388,
304-308[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Derynck, R.,
Zhang, Y.,
and Feng, X. H.
(1998)
Cell
95,
737-740[Medline]
[Order article via Infotrieve]
|
26.
|
Blottner, D.,
Wolf, N.,
Lachmund, A.,
Flanders, K. C.,
and Unsicker, K.
(1996)
Eur. J. Neurosci.
8,
202-210[Medline]
[Order article via Infotrieve]
|
27.
|
Ren, R. F.,
and Flanders, K. C.
(1996)
Brain Res.
732,
16-24[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Krieglstein, K.,
Reuss, B.,
Maysinger, D.,
and Unsicker, K.
(1998)
Eur. J. Neurosci.
10,
2746-2750[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Poulsen, K. T.,
Armanini, M. P.,
Klein, R. D.,
Hynes, M. A.,
Phillips, H. S.,
and Rosenthal, A.
(1994)
Neuron
13,
1245-1252[Medline]
[Order article via Infotrieve]
|
30.
|
Magal, E.,
Louis, J. C.,
Oudega, M.,
and Varon, S.
(1993)
Neuroreport
4,
779-782[Medline]
[Order article via Infotrieve]
|
31.
|
Sendtner, M.,
Arakawa, Y.,
Stockli, K. A.,
Kreutzberg, G. W.,
and Thoenen, H.
(1991)
J. Cell Sci. (Suppl.)
15,
103-109[Medline]
[Order article via Infotrieve]
|
32.
|
Richardson, P. M.
(1994)
Pharmacol. Ther.
63,
187-198[Medline]
[Order article via Infotrieve]
|
33.
|
Krieglstein, K.,
Farkas, L.,
and Unsicker, K.
(1998)
J. Neurobiol.
37,
563-572[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Symes, A. J.,
Pitts, R. L.,
Conover, J.,
Kos, K.,
and Coulombe, J.
(2000)
Mol. Endocrinol.
14,
429-439[Abstract/Free Full Text]
|
35.
|
Symes, A. J.,
Lewis, S. E.,
Corpus, L.,
Rajan, P.,
Hyman, S. E.,
and Fink, J. S.
(1994)
Mol. Endocrinol.
8,
1750-1763[Abstract]
|
36.
|
Jones, E. A.,
Conover, J.,
and Symes, A. J.
(2000)
J. Biol. Chem.
275,
36013-36020[Abstract/Free Full Text]
|
37.
|
de Caestecker, M. P.,
Parks, W. T.,
Frank, C. J.,
Castagnino, P.,
Bottaro, D. P.,
Roberts, A. B.,
and Lechleider, R. J.
(1998)
Genes Dev.
12,
1587-1592[Abstract/Free Full Text]
|
38.
|
Ho, S. N.,
Hunt, H. D.,
Horton, R. M.,
Pullen, J. K.,
and Pease, L. R.
(1989)
Gene (Amst.)
77,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Zawel, L.,
Dai, J. L.,
Buckhaults, P.,
Zhou, S.,
Kinzler, K. W.,
Vogelstein, B.,
and Kern, S. E.
(1998)
Mol. Cell
1,
611-617[Medline]
[Order article via Infotrieve]
|
40.
|
Symes, A. J.,
Rao, M. S.,
Lewis, S. E.,
Landis, S. C.,
Hyman, S. E.,
and Fink, J. S.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
572-576[Abstract]
|
41.
|
Brasier, A. R.,
Tate, J. E.,
and Habener, J. F.
(1989)
BioTechniques
7,
1116-1122[Medline]
[Order article via Infotrieve]
|
42.
|
Symes, A. J.,
Rajan, P.,
Corpus, L.,
and Fink, J. S.
(1995)
J. Biol. Chem.
270,
8068-8075[Abstract/Free Full Text]
|
43.
|
Tsukada, T.,
Horovitch, S. J.,
Montminy, M.,
Mandel, G.,
and Goodman, R. H.
(1985)
DNA
4,
293-300[Medline]
[Order article via Infotrieve]
|
44.
|
Danielson, P. E.,
Forss-Petter, S.,
Brow, M. A.,
Cavaletta, L.,
Douglass, J.,
Milner, R. J.,
and Sutcliffe, J. G.
(1988)
DNA
7,
261-267[Medline]
[Order article via Infotrieve]
|
45.
|
Dennler, S.,
Itoh, S.,
Vivien, D.,
ten Dijke, P.,
Huet, S.,
and Gauthier, J. M.
(1998)
EMBO J.
17,
3091-3100[Abstract/Free Full Text]
|
46.
|
Kretzschmar, M.,
Doody, J.,
Timokhina, I.,
and Massague, J.
(1999)
Genes Dev.
13,
804-816[Abstract/Free Full Text]
|
47.
|
de Caestecker, M. P.,
Yahata, T.,
Wang, D.,
Parks, W. T.,
Huang, S.,
Hill, C. S.,
Shioda, T.,
Roberts, A. B.,
and Lechleider, R. J.
(2000)
J. Biol. Chem.
275,
2115-2122[Abstract/Free Full Text]
|
48.
|
Jonk, L. J.,
Itoh, S.,
Heldin, C. H.,
ten Dijke, P.,
and Kruijer, W.
(1998)
J. Biol. Chem.
273,
21145-21152[Abstract/Free Full Text]
|
49.
|
Shi, Y.,
Wang, Y. F.,
Jayaraman, L.,
Yang, H.,
Massague, J.,
and Pavletich, N. P.
(1998)
Cell
94,
585-594[Medline]
[Order article via Infotrieve]
|
50.
|
Johnson, K.,
Kirkpatrick, H.,
Comer, A.,
Hoffmann, F. M.,
and Laughon, A.
(1999)
J. Biol. Chem.
274,
20709-20716[Abstract/Free Full Text]
|
51.
|
Attisano, L.,
and Wrana, J. L.
(2000)
Curr. Opin. Cell Biol.
12,
235-243[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Qing, J.,
Zhang, Y.,
and Derynck, R.
(2000)
J. Biol. Chem.
275,
38802-38812[Abstract/Free Full Text]
|
53.
|
Bannister, A. J.,
and Kouzarides, T.
(1995)
EMBO J.
14,
4758-4762[Abstract]
|
54.
|
Feng, X. H.,
Zhang, Y.,
Wu, R. Y.,
and Derynck, R.
(1998)
Genes Dev.
12,
2153-2163[Abstract/Free Full Text]
|
55.
|
Horvai, A. E.,
Xu, L.,
Korzus, E.,
Brard, G.,
Kalafus, D.,
Mullen, T. M.,
Rose, D. W.,
Rosenfeld, M. G.,
and Glass, C. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1074-1079[Abstract/Free Full Text]
|
56.
|
Janknecht, R.,
Wells, N. J.,
and Hunter, T.
(1998)
Genes Dev.
12,
2114-2119[Abstract/Free Full Text]
|
57.
|
Nakashima, K.,
Yanagisawa, M.,
Arakawa, H.,
Kimura, N.,
Hisatsune, T.,
Kawabata, M.,
Miyazono, K.,
and Taga, T.
(1999)
Science
284,
479-482[Abstract/Free Full Text]
|
58.
|
Bottner, M.,
Krieglstein, K.,
and Unsicker, K.
(2000)
J. Neurochem.
75,
2227-2240[CrossRef][Medline]
[Order article via Infotrieve]
|
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