Y-box-binding Protein YB-1 Mediates Transcriptional Repression of
Human
2(I) Collagen Gene Expression by Interferon-
*
Kiyoshi
Higashi
§,
Yutaka
Inagaki¶,
Noriyuki
Suzuki
,
Shinichi
Mitsui
,
Alain
Mauviel**,
Hideo
Kaneko
, and
Iwao
Nakatsuka
From the
Environmental Health Science Laboratory,
Sumitomo Chemical Company, Ltd., Konohana-ku, Osaka 554-8558, Japan, the ¶ Department of Community Health, Tokai University
School of Medicine, Isehara, Kanagawa 259-1193, Japan, the
Department of Cell Biology, Research Institute for Neuronal
Diseases and Geriatrics, Kyoto Prefectural University of Medicine,
Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan, and
** INSERM U532, Hôpital Saint-Louis, Paris
75010, France
Received for publication, August 26, 2002, and in revised form, November 13, 2002
 |
ABSTRACT |
We have demonstrated previously that a proximal
element within the human
2(I) collagen gene (COL1A2)
promoter mediates transcriptional repression by interferon-
(IFN-
), and designated this region the IFN-
response element
(IgRE). Screening of a human fibroblast cDNA expression library
with a radiolabeled IgRE probe exclusively yielded clones with a
sequence identical to that of the transcription factor YB-1.
Electrophoretic mobility shift assays (EMSA) using various IgRE-derived
oligonucleotide probes containing serial two-base mutations showed that
YB-1 protein was preferentially bound to the pyrimidine-rich sequence
within the IgRE. This region is located immediately downstream of and
partly overlaps the previously reported Sp1/Sp3 binding site.
Overexpression of YB-1 in human dermal fibroblasts decreased steady
state levels of COL1A2 mRNA and repressed
COL1A2 promoter activity in a dose-dependent
manner. This inhibitory effect of YB-1 on COL1A2 expression
was abolished by mutations of the IgRE shown to prevent YB-1 binding in
EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-
, suggesting that YB-1 mediates the inhibitory action of
IFN-
on COL1A2 promoter through its binding to the IgRE.
Also, overexpression of a deletion mutant YB-1, which lacks the
carboxyl-terminal domain, abrogated the repression of
COL1A2 transcription by IFN-
. A functional correlation
between IFN-
and YB-1 was further supported by luciferase assays
using four tandem repeats of the Y-box consensus oligonucleotide linked
to a minimal promoter. EMSA and Western blot analysis using cytoplasmic
and nuclear proteins implied that IFN-
promotes the nuclear
translocation of YB-1. Direct evidence for the nuclear translocation of
YB-1 by IFN-
was further provided by using a YB-1-green fluorescent
protein expression plasmid transfected into human fibroblasts.
Altogether, this study represents the definitive identification
of the transcription factor responsible for IFN-
-elicited inhibition
of COL1A2 expression, namely YB-1.
 |
INTRODUCTION |
Regulation of connective tissue formation is under rigorous
control by cytokines, which act in concert to ensure tissue integrity during homeostasis, development and repair (1). These cytokines have
been shown to control connective tissue cell recruitment and
proliferation, as well as synthesis and degradation of the extracellular matrix components (2). Disruption of this equilibrium, leading to excessive collagen deposition, is the hallmark of
interstitial fibrotic diseases. Type I collagen mRNA levels are
increased in experimental and clinical fibrotic states (3, 4), which implies a regulation at the transcriptional level. The role of transforming growth factor-
(TGF-
)1 as the principal
factor inducing collagen gene expression and leading to tissue fibrosis
has been suggested by the observations that (a) TGF-
expression often parallels increased type I collagen gene expression
(4, 5), and (b) a soluble TGF-
receptor prevents collagen
accumulation in experimental fibrosis (6).
Several studies have previously indicated possible mechanisms by which
tumor necrosis factor-
inhibits
2(I) collagen gene (COL1A2) expression at the transcriptional level (7-9).
Tumor necrosis factor-
has also been demonstrated to antagonize the effects of TGF-
through induction of inhibitory Smad7 (10) or AP-1
components (11). In contrast, identification of the transcription
factors involved in interferon-
-elicited down-regulation of
COL1A2 expression has been elusive, except that a cross-talk between TGF-
/Smad and IFN-
/Stat1 signaling pathways has recently been implicated in antagonistic regulation of gene transcription (12).
We have previously identified a proximal element within the human
COL1A2 promoter, spanning nucleotide
161 to
150, that mediates transcriptional repression by IFN-
. We designated this region the IFN-
response element (IgRE) (13). UV cross-linking experiments using nuclear extracts prepared from IFN-
-treated fibroblast cultures indicated that two DNA-protein complexes were formed with the IgRE (13). Interestingly, others have shown that Sp1
and Sp3 bind to a TCCCCC motif located between
164 and
159 (14),
immediately upstream of and partly overlapping the IgRE.
In the present study, we attempted to characterize the human fibroblast
nuclear protein that interacts with the IgRE by screening of a human
fibroblast cDNA expression library. We have identified the
transcription factor YB-1 as a component of the COL1A2
transrepressing complex bound to the IgRE. Cotransfection studies using
a YB-1 expression vector and reporter constructs containing the
wild-type and mutated COL1A2 IgRE have confirmed the role of YB-1 as a
negative regulator of COL1A2 transcription mediating the
effect of IFN-
.
 |
EXPERIMENTAL PROCEDURES |
Screening of cDNA Expression Library--
A
gt 11 human
dermal fibroblast cDNA library (Clontech) was
screened by Southwestern blotting using the 32P-labeled
three repeats of the IgRE oligonucleotide spanning
161 to
150 of
the human COL1A2 promoter as described previously (15). The
filters were subjected to a cycle of denaturation with 6 M guanidine hydrochloride followed by renaturation before screening. After incubation with 5% skim milk in the binding buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 10 mM MgC12, 1 mM EDTA, 1 mM dithiothreitol) for 30 min at 4 °C, the filters were
probed in the binding buffer containing 106 cpm/ml probe,
10 µg/ml denatured salmon sperm DNA, and 0.25% powdered milk. The
positive clones were sequenced directly using
gt 11 primers (Takara
Biomedicals, Kyoto, Japan).
Plasmids--
Point mutations were introduced into the YB-1
binding sites using mutational polymerase chain reaction (PCR) as
described previously (16, 17). The constructs
161M1/luc
and
161M5/luc containing two-base pair substitution
mutations (5'-CCCATTCGCTCC-3' to 5'-CGGATTCGCTCC-3' and
5'-CCCATTCGCGGC-3', respectively) were prepared by inserting the
mutated promoter sequences into a firefly luciferase gene vector, pGL3
basic vector (Promega, Madison, WI). Four copies of the Y-box consensus
oligonucleotide (CTGATTGGCTAA) linked to a minimal promoter containing
only a TATA box were cloned into pGL3 basic vector. A YB-1 expression
plasmid, YB-1/RSV, was constructed by ligating the entire YB-1 coding
sequence into the HindIII/XbaI sites of pRc/RSV
vector (Invitrogen). A deletion mutant YB-1 expression plasmid,
which lacks the carboxyl terminus, was constructed by ligating the
corresponding sequences into the BamHI/XhoI sites
of pcDNA3.1(+) vector (Invitrogen). The pCMX-YB-1-GFP was prepared
by ligating the YB-1 sequence into the
HindIII/PmlI sites of pCMX-hGR-GFP (kindly
provided by Dr. H. Ogawa, Kyoto University, Kyoto, Japan) (18). The
sequences of all plasmids were verified by automated sequencing
(Applied Biosystems, Foster City, CA).
Cell Culture and Transient Transfection--
Normal human dermal
fibroblasts (Clontech, Palo Alto, CA) were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (FCS). Transient transfections were performed using the
Lipofectin reagent (Invitrogen) according to the manufacturer's protocol. Forty hours after transfection, the cells were rinsed twice
with phosphate-buffered saline, harvested by scraping, and lysed in
lysis buffer (Promega). Aliquots containing the identical amounts of
protein, as measured with a commercial assay kit (BioRad), were
subjected to luciferase assays.
Real-time RT-PCR Assay--
Total RNA was isolated using Trizol
reagent (Invitrogen) according to the manufacturer's protocol. Fifty
nanograms of total RNA was reverse-transcribed using ImProm-II reverse
transcriptase (Promega). A pair of gene-specific PCR oligonucleotide
primers and an oligonucleotide probe labeled with a reporter
fluorescent dye at the 5'-end and a quencher fluorescent dye at the
3'-end were designed according to the guidelines suggested in the
TaqMan model 5700 sequence detection instrument manual (Applied
Biosystems). The human COL1A2 primers and probe used are as
follows: forward primer, 5'-CCAGAGTGGAGCAGTGGTTACTACT-3';
reverse primer, 5'-TTCTTGGCTGGGATGTTTTCA-3'; and probe,
5'-CTACTGGCGAAACCTGTATCCGGGC-3'. The human YB-1 primers and probe used
are as follows: forward primer, 5'-TCGCCAAAGACAGCCTAGAGA-3'; reverse
primer, 5'-TCTGCGTCGGTAATTGAAGTTG-3'; and probe,
5'-TCAGCAGCCACCTCAACGTCGGTA-3'. Human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene primer and probe mixture from the
predeveloped TaqMan assay reagents (Applied Biosystems) was
used. The thermal cycling condition included 1 cycle at 50 °C for 5 min, 1 cycle at 95 °C for 10 min, and 40 cycles at 95 °C for
15 s and at 60 °C for 1 min. Standard curves for expression of
each gene were generated by serial dilution of cDNA prepared from
human fibroblasts. The relative mRNA expression levels of
COL1A2 and YB-1 genes were normalized against those of
GAPDH gene in the same RNA preparation.
Expression and Purification of Recombinant
Protein--
Recombinant YB-1 protein was produced using the pET
system (Novagen, Madison, WI) as described previously (19). Briefly, after transformation of Escherichia coli BL21(DE3) with
YB-1/pET-28a(+) expression vector,
isopropyl-
-D-thiogalactopyranoside was added to LB
medium at 0.5 A600 followed by incubation
for another 3 h. The induced cells were collected and sonicated
until no longer viscous. The supernatant was applied to a
nickel-nitrilotriacetic acid-agarose column (Qiagen, Hilden,
Germany), and recombinant (His)6-YB-1 was eluted with
the buffer containing 200 mM imidazole. Purity of the
expressed YB-1 fusion product was ascertained by analytic SDS
polyacrylamide gel electrophoresis (PAGE).
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear and
cytoplasmic extracts were prepared according to the method of Andrews
and Faller (20). For EMSA, the probes (~50,000 cpm) were incubated
with recombinant YB-1 protein or nuclear extracts for 30 min on ice in
20 µl of binding reaction buffer as described previously (21). For
competition experiments, 20-500-fold molar excess of unlabeled IgRE or
consensus sequences for YB-1 (22) and Sp1/Sp3 (23) were added to the
binding reaction. In some experiments, antibody interference assays
were performed by preincubating nuclear extracts with 2 µg of
anti-YB-1 polyclonal antibodies prepared against the 15-amino acid
peptide (residues 299-313) of YB-1 as previously reported (24) or with
anti-Sp1 and anti-Sp3 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Western Blot Analysis--
Twenty micrograms of nuclear proteins
or 40 µg of cytoplasmic proteins were separated on a 7.5%
SDS-polyacrylamide gel, electroblotted, and incubated with anti-YB-1
antibodies. ECL detection system (Amersham Biosciences) was used to
detect immunoreactive proteins.
Fluorescence Microscopy--
Thirty-six hours after transfection
with 1 µg of pCMV-YB-1-GFP using the LipofectAMINE Plus reagent
(Invitrogen), 100 units/ml IFN-
was added into the culture
medium and incubated for another 6 h. After removing the
media, cells were washed with phosphate-buffered saline and examined
under a microscope (Nikkon, Tokyo, Japan) equipped with a
fluorescein isothiocyanate filter set for fluorescence detection
(18).
Statistical Analysis--
Values were expressed as mean ± S.D. Student's t test was used to evaluate the statistical
differences between groups, and a p value of less than 0.05 was considered significant.
 |
RESULTS |
Isolation of YB-1 by Southwestern Screening of a Human Dermal
Fibroblast cDNA Expression Library--
A human dermal fibroblast
cDNA library cloned into
gt 11 was screened using three tandem
repeats of the IgRE (
161 to
150) as a probe. After screening
~5 × 106 plaques, we obtained three positive
clones. Direct sequence analyses of the three clones revealed
overlapping subregions of the cDNA encoding YB-1, a member of the
Y-box transcription factor family (data not shown). The
COL1A2 IgRE sequence aligned in 7 of 12 bases with the
consensus Y-box sequence (Fig.
1A).

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Fig. 1.
Isolation of YB-1 by Southwestern screening
of a human dermal fibroblast cDNA expression library.
A, comparison between the IgRE of human COL1A2
and the Y-box consensus sequence. The COL1A2 IgRE sequence
aligns in 7 of 12 bases with the consensus Y-box sequence. The
identical bases are underlined. B, sequences of a
series of mutant oligonucleotides. WT indicates the
wild-type sequence. M1-M5 contains the indicated serial
two-base substitutions introduced into the IgRE sequence spanning 161
to 125 of the COLlA2 promoter. C,
identification of the essential bases for YB-1 binding to the IgRE.
Recombinant YB-1 protein (200 ng) was incubated with double-stranded
(ds), sense single-stranded (ss-S), or antisense
single-stranded (ss-AS) oligonucleotides as probes
(~50,000 cpm) at 4 °C for 30 min. Note that both the
-160C 159C and
-155T 154C bases were highly essential for
YB-1 binding to the IgRE.
|
|
Recombinant YB-1 Binds to the IgRE--
Although YB-1 was
identified originally as a transcription factor bound specifically to
the Y-box sequence containing a CCAAT motif (25), it has also been
shown to bind to pyrimidine-rich oligonucleotides that can adopt an
intramolecular triplex, single-stranded structure (26, 27). The IgRE
within the human COL1A2 promoter possesses both the
Y-box-like element and the pyrimidine-rich sequence (Fig.
1A). To examine the binding specificity of YB-1 to the IgRE,
a (His)6-YB-1 fusion protein was expressed in E. coli and used for gel mobility shift assays. Various
double-stranded oligonucleotides containing serial two-base mutations
or single-stranded oligonucleotides were used as probes to determine
the recognition targets of YB-1 (Fig. 1B). As shown in Fig.
1C, recombinant YB-1 effectively bound to both the
double-stranded (ds WT) and the sense single-stranded
(ss-S WT) IgRE, whereas complex formation was not observed
between YB-1 and the antisense single-stranded (ss-AS WT)
IgRE. To further characterize DNA sequences involved in complex
formation between YB-1 and double-stranded IgRE, a series of
oligonucleotides containing substitution mutations were used as probes
in EMSA. As shown in Fig. 1C, M1 and M5 oligonucleotide probes completely failed to form the YB-1·DNA complexes, and
M3 and M4 probes showed markedly diminished complex formation. In contrast, introduction of mutations into
-158A
157T (M2) had no effect on the binding
of YB-1 to the IgRE. These results suggest that both
-160C
159C and
-152T
151C bases were most essential for YB-1
binding to the IgRE.
Binding of Endogenous YB-1 and Sp1/Sp3 to the
IgRE--
To determine whether endogenous YB-1 binds to the IgRE, we
next performed EMSA using nuclear extracts prepared from human fibroblasts. As shown in Fig.
2A, incubation of nuclear
extracts with double-stranded IgRE probe yielded at least four retarded bands (arrows 1-4), all of which were diminished
by the addition of increasing amounts of unlabeled double-stranded
competitor. Interestingly, although formation of the three slowly
migrating complexes (arrows 1-3) was completely
abolished by adding a 20-fold molar excess of unlabeled double-stranded
competitor, the faster migrating complex (arrow
4) was still observed even after adding a 500-fold molar
excess of the competitor. These results suggested that the binding
affinities of those nuclear proteins to the IgRE are different from
each other. In addition, unlabeled sense single-stranded IgRE
interfered with the formation of complex 4 in a
dose-dependent manner (Fig. 2A), whereas it did
not affect complexes 1-3. On the other hand, antisense single-stranded
IgRE as a competitor failed to diminish the formation of complex 4 (data not shown). These results clearly demonstrated that the nuclear
factor(s) forming complex 4 preferentially bind to both the double- and the sense single-stranded IgRE. Considering the results shown in Fig.
1C, we then tested the possibility that YB-1 interacts with
the IgRE to form complex 4. For this purpose, we generated anti-YB-1
antibodies against the carboxyl terminus of YB-1, which inhibited the
binding of recombinant YB-1 to the IgRE (Fig. 2B). Preincubation of nuclear extracts with anti-YB-1 antibodies resulted in
significant but partial inhibition of the complex 4 formation (Fig.
2B). With regard to the slowly migrating complexes 1-3, a
previous study indicated the binding of Sp1/Sp3 to the
164 to
159
COL1A2 sequence, immediately upstream of and partly
overlapping the IgRE (23). Consistent with their results, the formation of complex 1 was interfered with by anti-Sp1 antibodies and that of complexes 2 and 3 was abolished by adding anti-Sp3 antibodies (Fig.
2B). Furthermore, in agreement with the results of Fig. 1C, M1 and M5 oligonucleotides as a probe hardly formed the
YB-1·IgRE complex (Fig. 2C). Interestingly, interruption
of YB-1 binding to the IgRE augmented the binding of Sp1 and Sp3 to the
IgRE (Fig. 2C).

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Fig. 2.
Binding of endogenous YB-1 and Sp1/Sp3 to the
IgRE. A, EMSA with oligonucleotide competitors. The
wild-type IgRE oligonucleotide as a probe (~50,000 cpm) was incubated
with 10 µg of nuclear extracts prepared from human dermal fibroblasts
in the presence of increasing amounts of unlabeled double-stranded
(ds) or sense single-stranded (ss-S) IgRE
oligonucleotide competitors (20, 100, and 500× for double-stranded; 20 and 100× for sense single-stranded). Specific DNA-protein complexes
are indicated by arrows. B, EMSA with antibodies.
The wild-type IgRE oligonucleotide as a probe was incubated with 200 ng
of recombinant YB-1 (rYB-1) or 10 µg of nuclear extracts
(NE) prepared from human dermal fibroblasts in the presence
of 2 µg of control IgG or specific antibodies, indicated at the
top of each lane. Specific DNA-protein complexes are
indicated by arrows. C, EMSA with mutant probes.
The wild-type or mutant IgRE oligonucleotide used as a probe was
incubated with 10 µg of nuclear extracts prepared from human dermal
fibroblasts. Specific DNA-protein complexes are indicated by
arrows. Note that substitution mutations introduced into the
-160C 159C and
-155T 154C bases most effectively diminished
YB-1 binding to the IgRE.
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|
YB-1 Suppresses COL1A2 Promoter Activity via the IgRE--
To
confirm the transcriptional repression of COL1A2 expression
by YB-1, we first performed real-time RT-PCR assays using Taqman probes. As shown in Fig. 3A,
expression of YB-1 suppressed endogenous COL1A2 mRNA
levels in a dose-dependent manner. We then examined whether
the binding of YB-1 to the IgRE is necessary for the regulation of
COL1A2 promoter activity. To this end, we transfected human dermal fibroblasts with either the wild-type
161WT or with mutated
161M1 and
161M5 reporter plasmids together
with a YB-1 expression plasmid. The basal transcription level of
161M1 was significantly higher than that of
161WT,
whereas the basal transcription level of
161M5 was not
statistically different from that of
161WT (Fig. 3B).
Overexpression of YB-1 significantly decreased transcription of
161WT
construct by about 50% in human dermal fibroblasts. In contrast, the
promoter activities of both the
161M1 and
161M5 constructs, which lack the YB-1 binding sequence
within the IgRE, were not affected by overexpression of YB-1 (Fig.
3B). Taken together, these results suggest that YB-1 is able
to suppress COL1A2 gene expression and that the inhibitory
action is exerted via a promoter region identified previously as the
IgRE (13).

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Fig. 3.
Both YB-1 and IFN-
suppress COL1A2 promoter activity via the YB-1
binding site. A, human dermal fibroblasts were
transfected with the indicated amounts of YB-1 expression vector. Six
hours after transfection, the cells were placed in medium supplemented
with 10% FCS and incubated for another 48 h. Total RNA (50 ng)
was extracted and quantitatively analyzed for COL1A2 ( )
or YB-1 ( ) mRNA expression using real-time RT-PCR. In all cases,
pRc/RSV control vector was used to ensure an equal amount of DNA in
each sample. The relative levels of COL1A2 or YB-1 gene
expression were normalized against the GAPDH levels measured in the
same total RNA preparation (mean ± S.D., n = 6).
B, the 161WT reporter plasmid was subjected to a
substitution mutation at either -160C 159C
( 161M1) or
-155T 154C ( 161M5)
within the IgRE in the same way as designed for the EMSA shown in Fig.
1B. Human dermal fibroblasts were transfected with 4 µg of
reporter plasmid together with 100 ng of either pRc/RSV (from
open bar to closed bar) or YB-1/RSV (from
closed bar to open bar) expression vector. Six
hours after transfection, the cells were placed in medium supplemented
with 10% FCS and incubated for another 40 h. The luciferase
activities, normalized by protein concentrations, were expressed in
relative luminescence units (mean ± S.D., n = 6).
C, human dermal fibroblasts were incubated with different
concentrations of IFN- (0-100 units/ml) for 48 h. Total RNA
(50 ng) was extracted and quantitatively analyzed for COL1A2
( ) or YB-1 ( ) mRNA expression using real-time RT-PCR. The
relative levels of COL1A2 or YB-1 gene expression were
normalized against the GAPDH levels measured in the same
total RNA preparation (mean ± S.D., n = 6).
D, human dermal fibroblasts were transfected with 4 µg of
wild-type or mutated reporter plasmid. Six hours after transfection,
the cells were placed in medium supplemented with 10% FCS. One hour
later, they were left untreated (from open bar to
closed bar) or treated (from closed bar to
open bar) with IFN- (1 unit/ml) and incubated for another
40 h. The luciferase activities, normalized by protein
concentrations, were expressed in relative luminescence units
(mean ± S.D., n = 6). E, human dermal
fibroblasts were transfected with 4 µg of 161WT reporter plasmid
together with 100 ng of pcDNA3.1(+) vector as control or with
expression plasmids encoding either full-length YB-1 or deletion mutant
YB-1 (N+CSD; corresponding to amino acids 1-129), which
lacks the carboxyl-terminal domain. Six hours after transfection, the
cells were placed in medium supplemented with 10% FCS. One hour later,
they were left untreated (from open bar to closed
bar) or treated (from closed bar to open
bar) with IFN- (1 unit/ml) and incubated for another 40 h.
The luciferase activities, normalized by protein concentrations, were
expressed in relative luminescence units (mean ± S.D.,
n = 6). An asterisk signifies that the
values are significantly different compared with control.
N.S., not significant.
|
|
YB-1 Mediates IFN-
-elicited Repression of COL1A2
Transcription--
The results described above led us to investigate
the role of YB-1 in mediating the inhibitory effect of IFN-
on
COL1A2 promoter activity. We first performed real-time
RT-PCR assays to confirm the transcriptional repression of
COL1A2 by IFN-
. As shown in Fig. 3C, IFN-
significantly suppressed endogenous COL1A2 mRNA levels
in a dose-dependent manner. On the other hand, the levels of endogenous YB-1 mRNA remained unchanged, indicating that
IFN-
-elicited inhibition of COL1A2 transcription is
independent of de novo synthesis of YB-1. Then, the mutated
constructs
161M1 and
161M5, as well as
their wild-type counterpart
161WT, were used in transient transfection experiments. Consistent with the results of our previous study (13), IFN-
exerted a significant transcriptional inhibition of
the wild-type
161WT construct by about 50%. In contrast, the point
mutations introduced into both -160C
159C
nucleotides (-161M1) and
-155T
154C nucleotides (-161M5)
prevented the COL1A2 response to IFN-
(Fig.
3D). These results clearly demonstrate that the YB-1 binding site within the 12-base-pair IgRE is essential for IFN-
responsiveness. To further confirm that YB-1 is involved in
COL1A2 repression by IFN-
, we generated a deletion mutant
YB-1 expression plasmid, which lacks the carboxyl-terminal region
compared with the full-length YB-1 expression plasmid. As shown
in Fig. 3E, transcriptional repression of COL1A2
by IFN-
was strengthened further by overexpression of YB-1. On the
other hand, overexpression of the mutant YB-1 abrogated the
COL1A2 repression by IFN-
while keeping the basal transcription levels unchanged (Fig. 3E). These results
clearly demonstrated that YB-1 not only regulates the COL1A2
transcription but also mediates IFN-
elicited repression of
COL1A2 transcription.
Y-box Consensus Sequence Confers IFN-
Responsiveness to a
Minimal Promoter--
To determine whether IFN-
exerts its
inhibitory effect solely through the YB-1 biding site or circumvents
any additional non-YB-1-specific cis-elements, we
constructed (YB-1)4-luciferase reporter plasmid in which
four tandem repeats of the consensus Y-box sequence were linked to a
minimal promoter containing only a TATA box. As shown in Fig.
4A, expression of YB-1
decreased (YB-1)4-luciferase activity in a
dose-dependent manner. Interestingly, a
dose-dependent diminution of (YB-1)4-luciferase
activity was also observed by changing the concentration of IFN-
in
culture medium (Fig. 4B). These data suggest that
IFN-
exerts its inhibitory action specifically through the Y-box
sequence.

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Fig. 4.
Y-box consensus sequence confers
IFN- responsiveness to a minimal
promoter. A, human dermal fibroblasts were transfected
with 4 µg of (YB-1)4-luciferase reporter gene plasmid
together with the different amounts of YB-1/RSV expression vector
(0-50 ng). The pRc/RSV vector DNA was used to maintain the equivalent
amount of transfected DNA in each dish. Six hours after transfection,
the cells were placed in medium supplemented with 10% FCS. Incubation
was continued for 40 h, and then reporter activity was
determined. B, human dermal fibroblasts were transfected
with 4 µg of (YB-1)4-luciferase reporter gene plasmid.
Six hours after transfection, the cells were placed in medium
supplemented with 10% FCS. One hour later, IFN- was added to the
medium at different concentrations (0-1 unit/ml) and incubated for
another 40 h. Luciferase activities, normalized by protein
concentration, were expressed in relative luminescence units (mean ± S.D., n = 6). An asterisk signifies that
the values are significantly different compared with control.
N.S., not significant.
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|
Nuclear Translocation of YB-1 by IFN-
--
The results
described above led us to investigate the IFN-
-induced alterations
in the relative amount and binding ability of YB-1 to the IgRE. We
first evaluated alterations in the relative amount of YB-1 in response
to IFN-
using Western blot analysis. Twenty micrograms of nuclear
proteins or 40 µg of cytoplasmic proteins prepared from cells either
untreated or treated with IFN-
for various lengths of time were
coelectrophoresed with recombinant YB-1. The amount of YB-1 present in
nuclear extracts was significantly increased by IFN-
treatment for
more than 4 h (Fig. 5A,
upper panel). Inversely, the amount of YB-1 present in
cytoplasmic extracts was obviously decreased (Fig. 5A,
lower panel). Then we analyzed IFN-
-induced alterations
in the binding ability of YB-1 to the IgRE using EMSA. As shown in Fig.
5B, the intensity of the YB-1·IgRE complex was increased
remarkably by IFN-
treatment. In contrast, the intensities of both
the Sp1·IgRE and Sp3·IgRE complexes remained unchanged (data not
shown). Furthermore, Southwestern blot analysis using IgRE as a probe
showed that the intensity of the band estimated at ~50 kDa, which is
almost identical to the size of YB-1, was significantly increased by
IFN-
treatment for more than 4 h (data not shown). These
results clearly demonstrated that IFN-
promotes the nuclear
translocation of YB-1 followed by its binding to the IgRE. To
demonstrate directly the nuclear translocation of YB-1 by IFN-
,
human dermal fibroblasts were transfected with 1 µg of YB-1-GFP
expression plasmid and treated with 100 units/ml IFN-
. YB-1-GFP
fusion protein was located mainly in the cytoplasm of untreated cells,
whereas nuclear translocation of YB-1-GFP was observed as early as
4 h after exposure to IFN-
(Fig.
6).

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Fig. 5.
IFN- increases YB-1
binding to the IgRE. A, Western blot analyses were
performed using nuclear (upper panel) and cytoplasmic
(lower panel) extracts prepared from human fibroblasts
untreated or treated with 100 units/ml IFN- for various lengths of
time as indicated, and recombinant YB-1 was electrophoresed in
parallel. Note that the intensity of the band corresponding to
YB-1 was increased by IFN- treatment. B, The wild-type
IgRE oligonucleotide as a probe was incubated with 10 µg of nuclear
extract prepared from cells either untreated or treated with IFN-
(100 units/ml) for various lengths of time as indicated. Note that the
intensity of the band corresponding to YB-1 was increased by IFN-
treatment.
|
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Fig. 6.
IFN- -induced nuclear
translocation of YB-1. Human dermal fibroblasts were
transiently transfected with 1 µg of pCMV-YB-1-GFP expression
plasmid. Six hours after transfection, the cells were placed in medium
supplemented with 10% FCS. After incubation for 40 h, the cells
were left untreated (A) or treated (B) with 100 units/ml IFN- for 4 h. Then the cells were examined under a
microscope equipped with a fluorescein isothiocyanate filter set for
fluorescence detection.
|
|
 |
DISCUSSION |
We have demonstrated previously that a proximal region of the
human COL1A2 promoter is essential for mediating the
inhibitory effect of IFN-
and designated this region the IFN-
response element, IgRE (13). In the current study, by screening
a human fibroblast cDNA expression library, we identified YB-1 as a
transcription factor that interacts with the IgRE. Both bacterially
expressed recombinant protein and endogenous YB-1 present in fibroblast nuclear extracts exhibited high affinity for the IgRE. Transient transfection assays demonstrated that expression of YB-1 decreased COL1A2 promoter activity in a dose-dependent
manner and that IFN-
suppressed gene transcription through YB-1
binding to the IgRE. Experiments using the carboxyl domain-deleted YB-1
confirmed the role of YB-1 in COL1A2 repression by IFN-
.
Furthermore, a combination of Western blot analysis, EMSA of nuclear
proteins, and GFP fluorescence study demonstrated that IFN-
treatment translocates YB-1 into the nucleus and increases the binding
of YB-1 to the IgRE sequence.
Ihn and co-workers (14, 23) have demonstrated that ubiquitous
transcription factors Sp1 and Sp3 bind to a TCCCCC motif located
between
164 and
159 within the human COL1A2 promoter. Using transient transfection assays, they also showed that this pyrimidine-rich motif is a binding site for a transcriptional repressor
(14, 23, 28). However, since the blockade of Sp1/Sp3 binding to the
repressor site did not affect the collagen promoter activity (23), the
functional role of Sp1/Sp3 interacting with the TCCCCC motif has not
been fully understood. Our present study has clearly indicated that the
IgRE (
161 to
150), immediately downstream of and partly overlapping
the Sp1/Sp3 binding site, is a binding site for a COL1A2
repressor, YB-1. Indeed, Ihn et al. (28) introduced a
substitution mutation into
161 CCC
159
within the TCCCCC motif, which was almost comparable with our M1
mutation, and this resulted in a 6-fold increase in the basal promoter activity.
Functional assays using four tandem repeats of the YB-1 consensus
sequence indicated that IFN-
exerts its inhibitory action on
COL1A2 transcription solely through the YB-1 binding site
(Fig. 4). It should be noted, however, that the binding of Sp1/Sp3 was markedly enhanced when using the mutated IgRE sequences (M1 and M5) as
EMSA probes (Fig. 2C). YB-1 has been shown to interact with
other transcription factors and viral proteins (29-31). In addition, a
previous study has suggested that the level of Sp1 activity dictates
binding of YB-1 to its target sequence and therefore affects its
regulatory function (32). Thus, it could be argued that YB-1 and
Sp1/Sp3 regulate COL1A2 transcription through their physical
interactions and/or competitive binding to the adjacent IgRE and the
TCCCCC motif. In contrast to the mutation introduced into
-160C
159C, the other substitution mutation
introduced into another essential YB-1 binding site
(-155T
154C) did not affect the basal
promoter activity (Fig. 3). We suggested previously (13) that a protein
with size estimated at ~30 kDa definitely interacts with the IgRE
through the -155T
154C nucleotides and acts
as a transactivator. It is therefore possible that the lack of binding
of both YB-1 and the 30-kDa protein did not affect promoter activity.
In addition, Southwestern blot analyses showed that IFN-
treatment
increased YB-1 binding to the IgRE, whereas it had no effect on the
binding of the 30-kDa protein (data not shown). Altogether, reciprocal
interactions between YB-1, Sp1/Sp3, and the 30-kDa protein might be
required for regulation of COL1A2 transcription by IFN-
.
The significant but partial inhibition of YB-1·IgRE complex formation
by adding anti-YB-1 antibodies (Fig. 2B) may indicate that
the 30-kDa protein interacts with YB-1 and interfere with its antibody
recognition. The nature of the 30-kDa protein as well as the mechanisms
of its functional interaction with YB-1 also remain to be elucidated.
It has been shown that chk-YB-1b, a chicken homologue of human YB-1,
binds to a pyrimidine-rich sequence and activates the rat
1(I)
procollagen gene (COL1A1) transcription (33). Amino acid
sequences of the nucleic acid binding domain and the carboxyl terminus
of chk-YB-1b display a high degree of homology with those of human
YB-1. In contrast, the sequence of the amino-terminal domain, which may
influence the ability of the protein to interact with other nucleic
acid binding proteins, is the most structurally distinct portion (34).
Norman et al. (35) have shown that YB-1 overexpression
suppresses endogenous COL1A1 expression and collagen protein
production in rodent cells. Sequence analysis of the mouse
COL1A1 promoter revealed three putative YB-1 binding sites,
83/-72,
103/-92, and
129/-118, all of which are well conserved
in human (35). In addition, Yuan et al. (36) have demonstrated transcriptional inhibition of human COL1A1
expression by IFN-
and located the IFN-
response element between
nucleotides
129 and
109 of the human COL1A1 promoter. In
the present study, we have shown that YB-1 suppresses transcription of
human COL1A2 gene and that IFN-
inhibits gene
transcription via the YB-1 binding site. Taken together, these results
suggest that IFN-
/YB-1 signaling may coordinately down-regulate both
COL1A1 and COL1A2 gene transcription.
Consensus sequence for translocation of proteins to the nucleus does
not exist in YB-1. However, nuclear translocation signals are located
in its carboxyl terminus, which typically contain a cluster of three to
six basic residues in a short peptide of four to nine amino acids (25).
In this study, deletion of the carboxyl-terminal domain of YB-1
containing those nuclear translocation signals resulted in an
abrogation of COL1A2 repression by IFN-
. Recently,
Stenina et al. (37) have suggested that thrombin induces the
release of YB-1 from mRNA by proteolytic cleavage and the truncated
YB-1 is translocated into the nucleus and bound to the thrombin
response element (CCACCCACC) in endothelial cells. However, Western
blot analyses using an amino-terminally Flag-tagged YB-1 expression
vector failed to show degradation of YB-1 following IFN-
treatment
of human fibroblasts (data not shown). In contrast, a previous study
using human cancer cells showed that the nuclear translocation of YB-1
is induced by UV irradiation or anticancer drugs like cisplatin in a
protein kinase C-dependent manner (38). Our results showed
that treatment with 100 nM
12-O-tetradecanoylphorbol-13-acetate also initiated
the nuclear translocation of YB-1 in human fibroblasts (data not
shown). These results may suggest that some of the IFN-
-induced biological events, such as the induction of YB-1 translocation or the
inhibition of type I collagen gene expression, are mediated through the
protein kinase C pathway (39, 40). The precise mechanism by which
IFN-
initiates the nuclear translocation of YB-1 is currently under investigation.
Altogether, this study represents the definitive identification of the
transcription factor responsible for IFN-
-elicited inhibition of
COL1A2 expression, namely YB-1. Future studies aimed at the
characterization of the molecular mechanism involved in the regulation
of type I collagen gene expression by YB-1 may provide a novel insight
into the interstitial fibrotic diseases and eventually contribute to
the development of useful therapeutic means.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hidesato Ogawa in Kyoto
University for the generous gift of pCMX-hGR-GFP plasmid. We also thank
Dr. Ko Fujimori for valuable comments on this study and Yukiko Miyama
for excellent technical assistance.
 |
FOOTNOTES |
*
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. Tel.: 81-6-6466-5306;
Fax: 81-6-6466-5319; E-mail: higashik2@sc.sumitomo-chem.co.jp.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208724200
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
COL1A1,
1(I) collagen
gene;
COL1A2,
2(I) collagen gene;
IFN-
, interferon-
;
IgRE, IFN-
response element;
FCS, fetal calf serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
EMSA, electrophoretic mobility shift assays;
GFP, green fluorescence protein;
CMV, cytomegalovirus;
RT-PCR, reverse transcriptase
PCR.
 |
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