From the Department of Medicine, Division of
Rheumatology and Immunology, and the ** Laboratory of Cancer Genomics,
Hollings Cancer Center, Medical University of South Carolina,
Charleston, South Carolina 29401 and the
Department of
Dermatology, Kanazawa University, Kanazawa, 920-0947 Japan
Received for publication, November 7, 2000, and in revised form, March 15, 2001
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ABSTRACT |
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Fibrosis is characterized by the excessive
deposition of extracellular matrix (ECM), especially collagen. Because
Ets factors are implicated in physiological and pathological ECM
remodeling, the aim of this study was to investigate the role of Ets
factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen ECM1 remodeling is a
complex and tightly regulated process that occurs during embryogenesis,
the female reproductive cycle, angiogenesis, and wound repair. However,
in the majority of normal adult tissues only limited turnover of the
ECM takes place. In contrast, in many pathological conditions the
balance between ECM synthesis and degradation is disrupted, leading to
abnormal ECM remodeling. Excessive ECM breakdown is associated with
rheumatoid arthritis, osteoarthritis and periodontitis as well as tumor
invasion and metastasis (1-4), whereas excessive ECM deposition occurs in fibrotic diseases such as scleroderma, liver cirrhosis, and glomerulosclerosis (5-7). The type I collagen, a product of two coordinately regulated genes, Ets proteins comprise a family of transcription factors with over 30 members identified to date that are characterized by the presence of a
highly conserved DNA binding domain termed the Ets domain (17). Ets
proteins are grouped into subfamilies based on the sequence similarity
of the Ets domain and its location within the protein as well as
additional sequence similarities (17). Fli-1 and the closely related
Erg are the members of the ERG subfamily. Extensive in vitro
studies as well as the data obtained from various Fli-1 null mice
support a crucial role for Fli-1 in hematopoiesis (18). Fli-1 is also
expressed in embryonic tissues at the sites of cell migration and
epithelial-mesenchymal transition, suggesting additional functions
during development (19). It may be relevant that Fli-1 is an inducer of
tenascin-C, an ECM molecule, which is also expressed at the sites of
cell migration (20). However, the specific role of Fli-1 in these processes is presently not well understood. Targeted disruption of the
Fli-1 gene resulted in hemorrhage into the neural tube and the
ventricles of the brain at E11, resulting in embryonic death shortly
thereafter. In addition to hematopoietic defects, these mutant embryos
demonstrated a disruption of basement membrane tissues (18).
There is increasing evidence that some Ets proteins, particularly the
members of Ets-1 and E1AF/PEA3 subfamilies, play an important role in
regulating transcriptional activation of ECM-degrading enzymes
including serine proteases and matrix metalloproteinases (MMPs) (21,
22). Extensive previous studies of the polyoma virus enhancer as well
as urokinase plasminogen activator and several MMP promoters have
established a paradigm for the role of Ets in the regulation of this
group of genes (23). These promoters contain functional Ets binding
site(s) (EBS(s)) and AP-1 site(s). The members of the Ets and Jun
families that bind to the Ets-AP-1 composite element are also targets
of the Ras-mitogen-activated protein kinase signaling pathway (24).
Thus, the transcriptional activation of this group of genes depends on
the nature of interacting proteins (both Ets and AP-1 are multiprotein
families) as well as the exogenous stimuli activating the Ras cascade
(25).
Based on our recent observation that a matrix protein, tenascin-C, is
also regulated by Ets factors (20), we postulated that the role of Ets
in ECM remodeling may not be limited to regulation of the degradative
pathways but may also include regulation of synthesis of the ECM
proteins (26). To test this hypothesis we selected a well characterized
system with physiological and pathological relevance, the human
COL1A2 gene. In the present study we demonstrate that the
collagen gene is indeed a target for Ets factors. Significantly, we
provide evidence that Fli-1 acts as a transcriptional repressor of this
gene, whereas Ets-1 acts as an activator. Furthermore, we show that
cellular context plays an important role in defining both the
magnitude and direction of response to Fli-1.
Cell Culture and Generation of Stable Transfectants--
Human
dermal fibroblast cultures were established from newborn foreskins
obtained from the delivery suits of local hospitals. Foreskin tissue
was dissociated enzymatically by 0.25% collagenase type I (Sigma) and
0.05% DNase (Sigma) in Dulbecco's modified Eagle's medium with 20%
fetal calf serum (Life Technologies, Inc.). Human mesangial cells were
purchased from Clonetics (Walkersville, MD). HepG2 and HeLa cells were
purchased from the American Type Culture Collection (Manassas, VA).
Fli-1 and control stable transfectants were generated in human foreskin
fibroblasts transfected by the calcium phosphate technique with either
pSG5 Fli-1 or pSG5 control vector and were grown in the presence of
100-200 µg/ml of geneticin (Life Technologies, Inc.). Individual
antibiotic-resistant colonies were expanded and analyzed for the
expression of Fli-1 protein.
Procollagen Analysis by [3H]Proline Incorporation,
SDS-Polyacrylamide Gel Electrophoresis, and Autoradiography--
The
analysis of labeled proline incorporation into secreted protein was
performed as described previously (27). Fibroblasts were plated in
12-well plates and grown to visual confluency. The medium was changed
to serum-free medium (Dulbecco's modified Eagle's medium)
supplemented with 50 mg/ml ascorbic acid and 64 mg/ml Western Blot--
To determine the levels of expression of Ets-1
and Fli-1 proteins, total cell lysates (100 µg/lane) were used. To
detect Ets-2 protein, nuclear extracts (35 µg/lane) were used.
Samples were electrophoresed in 12% SDS-polyacrylamide gel and
transblotted onto polyvinylidene difluoride membrane (Millipore). After
blocking with 5% milk, the membranes were incubated with primary
antibodies, followed by incubation with horseradish
peroxidase-conjugated secondary antibodies and washing with
Tris-buffered saline buffer. Immune complexes were detected using
enhanced chemiluminescence (Amersham Pharmacia Biotech). The monoclonal
anti-Ets-1 antibody was obtained from Transduction Laboratories, the
polyclonal anti-Ets-2 antibody was obtained from Santa Cruz
Biotechnology, and the polyclonal anti-Fli-1 antibody was described
previously (18).
Northern Blot--
Total RNA was extracted from confluent
fibroblasts and analyzed by Northern blotting as described previously
(27). Filters were sequentially hybridized with radioactive probes for
Plasmid Constructs--
The Cell Culture Transfection and Reporter Gene
Assays--
Transient transfections with the indicated reporter,
expression, and control constructs were performed in duplicate in
6-well plates using FuGene 6 reagent (Roche Molecular Biochemicals)
according to the manufacturer's specifications. Transfections were
repeated at least four times using two different plasmid preparations. CAT and luciferase activities in aliquots containing equal amounts of
protein were determined 48 h post-transfection.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared according to Andrews and Faller (29) with minor modifications.
In vitro transcribed and translated human Fli-1, Ets-1, and
Ets-2 proteins were prepared using the TNT coupled reticulocyte lysate
system (Promega).
DNA Binding Assays--
EMSAs were performed with
32P-labeled probes as described previously (30). Briefly, 7 µg of nuclear extracts were incubated for 30 min on ice in 24 µl of
the binding buffer (20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, 10 µg/ml
aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin) containing
50,000-cpm labeled probe and 2 µg of poly(dI-dC)-poly(dI-dC)
(Amersham Pharmacia Biotech). In some assays, double-stranded
competitors (50-fold molar excess) or antibodies were preincubated with
nuclear extracts 30 min prior to the addition of a radioactive
nucleotide probe. Polyclonal anti-Sp1 and anti-Sp3 antibodies were
purchased from Santa Cruz Biotechnology, and monoclonal anti-Ets-1 was
purchased from Transduction Laboratories. Polyclonal anti-Fli-1
antibody was characterized previously (18). The separation of free
radiolabeled DNA from DNA-protein complexes was carried out on a 5%
nondenaturing polyacrylamide gel. Electrophoresis was performed in
0.5× Tris borate electrophoresis buffer at 250 V at 4 °C. The gels
were dried and exposed to x-ray film at Statistical Analysis--
The Mann-Whitney U test or
Wilcoxon test was used to determine statistical significance.
Fli-1 Inhibits Production of Collagenous Proteins and mRNA in
Human Dermal Fibroblasts--
In the first set of experiments we asked
whether Fli-1 regulates ECM production by generating stable Fli-1
transfectants in human dermal fibroblasts. Several clones that
exhibited elevated levels of Fli-1 protein were selected for further
investigation (Fig. 1A).
Overexpression of Fli-1 in dermal fibroblasts did not affect their
proliferative rate (Fig. 1B). To assess the effect of Fli-1
on the production of collagenous proteins, conditioned medium from
cells metabolically labeled with [3H]proline was analyzed
by SDS-polyacrylamide gel electrophoresis. As shown in Fig.
1C, increased expression of Fli-1 protein inversely correlated with the production of collagenous proteins. These data
indicate that elevated expression of Fli-1 has a potent inhibitory effect on the newly synthesized collagenous proteins. Decreased levels
of the collagenous proteins in conditioned medium may result either
from the activation of the degradative pathways (e.g. MMPs), decreased collagen synthesis, or both. To determine whether Fli-1 directly affects collagen gene expression, COL1A2 mRNA levels were
compared in Fli-1 stable transfectants and control clones. As shown in
Fig. 1, D and E, Fli-1 decreased COL1A2 mRNA
levels by at least 1.5-fold, suggesting that its inhibitory effects
occurs at least partially via regulating collagen mRNA expression
levels.
Fli-1 Inhibits COL1A2 Promoter Activity--
The effect of Fli-1
on the activity of the COL1A2 promoter was examined next.
Consistent with the effect of Fli-1 on the COL1A2 mRNA levels,
Fli-1 inhibited COL1A2 promoter activity in a
dose-dependent manner (Fig.
2, A and B). To map
the Fli-1 response element in the COL1A2 promoter, we
utilized previously generated promoter deletion constructs (10). The
strongest inhibition (2.8-fold) was observed with the EBS 3 and Sp1/Sp3 Binding Sites Function as an Fli-1 Response
Element--
To determine which of the EBSs were responsible for
mediating the Fli-1 inhibition, the putative EBSs were mutated
individually and in combination (as described under "Materials and
Methods"; see Table I). The Fli-1 Does Not Compete with Sp1 for DNA Binding--
To further
examine the possible functional interactions between Fli-1 and Sp1/Sp3,
we first evaluated whether Fli-1 could directly interfere with Sp1/Sp3
DNA binding. To test this potential mechanism, we compared binding of
the Sp1 and Sp3 to the Fli-1 Interacts Directly and Indirectly with the COL1A2
Promoter--
To examine interaction of Fli-1 with the collagen
promoter, we used the
To determine the role of Sp1/Sp3 in Fli-1 interactions with the
COL1A2 promoter, we used as a probe the promoter fragment in
which all three Sp1 binding sites were mutated. As previously shown,
these mutations abolish Sp1/Sp3 binding (Fig. 5B, lane 5). Significantly, Fli-1 binding was also substantially decreased with this probe as indicated by diminished supershift with the anti-Fli-1 antibody (Fig. 5B, lane 6).
Quantitative analysis of six independent experiments shows a
statistically significant 25% (p < 0.05) decrease in
Fli-1 binding between the wild-type probe and the probe containing
mutations in Sp1/Sp3 sites. Taken together, these in vitro
binding data indicate that Fli-1 interacts with the COL1A2
promoter even in the absence of EBSs. On the other hand, the binding of
Sp1/Sp3 to this promoter region seems to facilitate Fli-1 binding.
To explore this dual mechanism further, we utilized two Fli-1 mutants:
an Fli/W321R mutant harboring a single amino acid mutation in the Ets
domain that abolishes its ability to bind
DNA2 (32) and a deletion
mutant containing only the Ets domain (Fli/DBD). The effects of
overexpression of these two Fli-1 mutants on the COL1A2
promoter were investigated (Fig. 6). In
comparison to the wild-type Fli-1, both mutants caused more modest but
consistently reproducible decreases in the COL1A2 promoter
activity, suggesting that direct (via DNA binding) and indirect (via
protein-protein interaction) mechanisms contribute to the effects of
Fli-1 on the COL1A2 promoter.
Ets-1and Fli-1 Have Antagonistic Effects on the COL1A2 Promoter and
Compete with Each Other in Regulation of the COL1A2 Promoter
Activity--
What is the molecular mechanism for the Fli-1-mediated
repression? Human dermal fibroblasts express several Ets factors
including Fli-1, Ets-1, and Ets-2 (Fig.
7A). In contrast to Fli-1,
Ets-1 and Ets-2 stimulated the COL1A2 promoter in dermal
fibroblasts. Ets-1 stimulated the COL1A2 promoter with
slightly higher potency than Ets-2 (data not shown) and was selected
for further investigation. Although we have previously shown that
GABP
To assess the possibility of competition between Ets-1 and Fli-1 for
the COL1A2 promoter, a constant amount of Fli-1 and the COL1A2/LUC was co-transfected with increasing concentrations
of Ets-1 into dermal fibroblasts (Fig. 7B). Ets-1 was able
to reverse the inhibitory effects of Fli-1 in a
dose-dependent manner, suggesting that these two factors
may compete for binding to the COL1A2 promoter. Consistent
with this model, Ets-1 binding to the COL1A2 promoter was
observed by EMSA; however, Ets-1 binding seemed to be less prominent
than that observed with Fli-1 (Fig. 5C).
To compare the binding affinity of Fli-1 and Ets-1 to the
COL1A2 promoter, the in vitro translated Ets-1
and Fli-1 were reacted with the short promoter fragments containing
individual EBSs in the presence or absence of the corresponding
antibodies. The Fli-1 protein did not react with any of the promoter
fragments (data not shown), whereas the Ets-1 bound weakly only to the
promoter fragment containing EBS 3 (data not shown). These data suggest that either Ets-1 or Fli-1 alone have low binding affinity for the
COL1A2 promoter. Thus, their presence in the DNA-protein
complexes most likely depends on the presence of other transcription
factors interacting with this promoter. As suggested previously, the
candidate cofactors may be Sp1 and Sp3.
Ets-1 and Fli-1 Differ in Their Ability to Functionally Interact
with Sp1/Sp3 in the Gal4 System--
To examine the interaction
between Sp1/Sp3 and Ets factors further, we utilized the Gal4 system.
Either Sp1 or Sp3 fused to the Gal4 DBD domain-transactivated
Gal4-responsive promoter in human fibroblasts (Fig.
8A). Interestingly, Sp3 was a
more potent activator than Sp1 in the context of the Gal4 system.
Co-transfection of Ets-1 with either Gal4-Sp1 or Gal4-Sp3 further
enhanced Gal4-responsive promoter activity (Fig. 8A). This
supports a model for functional interaction between Sp1/Sp3 and Ets-1
in dermal fibroblasts. Surprisingly, Fli-1 was also able to synergize
with the Gal4-Sp1 and Gal4-Sp3 chimeric proteins under these
experimental conditions. These observations, together with our previous
data using the tenascin-C promoter (20), suggest that Fli-1 can behave
either as a repressor or an activator depending on the promoter
context. In contrast to fibroblasts, in HepG2 cells, Fli-1 did not have
a significant effect on either Gal4-Sp1 or Gal4-Sp3 activation
potential, whereas Ets-1 synergized with both Sp1 and Sp3 in activating
the Gal4 promoter (Fig. 8B). These data suggest that cell
type-specific cofactors may be required to facilitate Fli-1
interactions with Sp1/Sp3. Consistent with this possibility, previous
studies have demonstrated that the formation of a ternary complex and
the functional interaction between Sp1 and Ets-1 required presence of
the Tax protein (33). The difference between Fli-1-Sp1 interaction
observed here further suggests that distinct cofactors facilitate Ets-1 and Fli-1 interaction with Sp1/Sp3. Together, these data suggest that
the repressor function of Fli-1 depends on cell and promoter context.
Effects of Fli-1 and Ets-1 on the COL1A2 Promoter Are Cell
Type-specific--
To further examine the variable responses of
different cell types to Fli-1, we utilized several cell lines of
different origins in the analyses of Ets-1 and Fli-1 regulation of the
COL1A2 promoter (Fig.
9A). Ets-1 stimulated the
COL1A2 promoter activity in all cell lines tested. In
contrast, Fli-1 showed cell line-specific effects. In human lung
fibroblasts, Fli-1 significantly inhibited the COL1A2
promoter. In human mesangial cells and in HepG2 cells, Fli-1 had a
slight but consistent inhibitory effect, whereas in HeLa cells, Fli-1
was stimulatory for the COL1A2 promoter. We have also
determined the endogenous expression of Ets-1 and Fli-1 proteins in the
cells used in this experiment. Ets-1 and Fli-1 were expressed in all
cell types and although there were some differences in expression
levels, the ratio of Ets-1 to Fli-1 protein seemed to be similar in all
cells (Fig. 9B).
The present study establishes for the first time that Ets factors
play a critical role in regulating the expression of the collagen type
I gene in dermal fibroblasts. The following observations support this
conclusion. First, the exogenous expression of Fli-1 in dermal
fibroblasts led to a dramatic decrease of the newly synthesized
collagenous proteins and the COL1A2 mRNA. Second, transient transfection experiments have defined a functional EBS in the
COL1A2 promoter. Third, binding assays have demonstrated that Fli-1 and Ets-1 form DNA-protein complexes with sequences present
in the COL1A2 promoter. Furthermore, this study has also begun to unravel specific regulatory mechanisms involved in the transcriptional control of the COL1A2 gene by Ets factors.
Experiments using Fli-1 dominant interference and DNA binding mutants
indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (protein-protein interaction) mechanisms. Based on the
functional studies with the Sp1/Sp3 promoter substitution mutants as
well as the EMSA results, we postulate that functional interaction of
Fli-1 with Sp1 and Sp3 is essential for the inhibitory function of
Fli-1. It also seems that additional tissue-specific bridging factors
are involved in these interactions. The nature of these cofactors is
presently unknown. Furthermore, the competition experiments suggest
that Fli-1 inhibits the COL1A2 promoter by displacing Ets-1.
Together, these findings imply that the ratio of Fli-1 to Ets-1 and the
presence of co-regulatory proteins may contribute to the control of
collagen production in fibroblasts.
Our findings suggest that in the context of the COL1A2
promoter, Sp1 may play a dual role of an activator and a repressor of
this promoter (Fig. 10). Moreover,
Fli-1 and a tissue-specific co-repressor(s) may contribute to formation
of this repressor complex. Recent observations indicating direct
interaction between Sp1 and histone deacetylase-1 further support this
possibility (34). A possible mechanism explaining such a dual role has
been proposed for a tumor suppressor protein, p53 (35, 36). It has been
demonstrated that p53-mediated repression of specific target genes
depends on association of p53 with histone deacetylase-1 and Sin 3A
(35), whereas the activator status of p53 can be enhanced through
acetylation by p300 (36). It is intriguing that in our system, Sp1 can
behave as an activator and a co-repressor in the context of the same
promoter. What determines the switch between these two states remains
to be elucidated. However, it has been shown recently that Sp1 is
involved in mediating TGF-2(I) (COL1A2) mRNA was
inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a
critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In
vitro binding assays demonstrate that both Fli-1 and Ets-1 form
DNA-protein complexes with sequences present in COL1A2 promoter.
Furthermore, Fli-1 binding to the COL1A2 is enhanced via
Sp1-dependent interaction. Studies using Fli-1 dominant
interference and DNA binding mutants indicate that Fli-1 inhibition is
mediated by both direct (DNA binding) and indirect (via protein-protein
interaction) mechanisms and that Sp1 is an important mediator of the
Fli-1 function. Furthermore, experiments using the Gal4 system in the
context of different cell types as well as experiments with the
COL1A2 promoter in different cell lines demonstrate that
the direction and magnitude of the effect of Fli-1 is promoter- and
cell context-specific. We propose that Fli-1 inhibits
COL1A2 promoter activity by competition with Ets-1. In
addition, we postulate that another factor (co-repressor) may be
required for maximal inhibition after recruitment to the Fli-1-Sp1
complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence
of co-regulatory proteins ultimately control ECM production in fibroblasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) and
2(I) (COL1A2), is a predominant ECM
component of the fibrotic lesion. Studies using diverse experimental systems have indicated that transcriptional regulation of the type I
collagen genes is species- and tissue-specific (5). The
COL1A2 promoter has been extensively used as an experimental model system to delineate transcriptional regulation of the collagen gene in human tissues. This promoter constitutively exhibits high levels of expression in activated dermal fibroblasts, which are "turned on" by routine in vitro tissue culture
conditions such as growth on plastic in the presence of serum. Previous
studies have identified the ubiquitous factors, Sp1 and Sp3, and CAAT binding factor as primary activators of this promoter (9, 10). In contrast, very little is currently known about the nature of the
transcriptional repressors that may counteract or modulate the potent
stimulatory effects of Sp1 and Sp3 on this promoter. The activity of
the COL1A2 promoter can be modulated further by transcriptional factors mediating cytokine responses. Thus, Smad3 in
cooperation with p300/CREB-binding protein mediates activation of this
promoter by TGF-
(8, 11-13), whereas CAAT/enhancer-binding protein
as well as nuclear factor-
B seem to be involved in tumor necrosis
factor-
inhibition of the COL1A2 activity (14, 15). Moreover, recent studies provide evidence for Sp1 as a critical mediator of TGF-
induction of this promoter via interaction with Smad3 (8, 16).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino
propiontrile for the duration of the experiment. After a 24-h
incubation in serum-free medium, 10 µCi/ml
L-[2,3,4,5-3H]proline (specific activity 3.6 TBq/mmol) (Amersham Pharmacia Biotech) was added for 24 h. The
medium was harvested from each well, and the cells were trypsinized and
counted. The medium was dehydrated in a SpeedVac (Savant) and
resuspended in SDS/dithiothreitol sample buffer and boiled to denature.
The volume of sample buffer added for resuspension was normalized
according to the measured cell count of each well. After
electrophoresis, gels were enhanced by immersion in 2,5-diphenyloxazole
and visualized by autoradiography.
2(I) procollagen and glyceraldehyde-3-phosphate dehydrogenase.
The filters were scanned with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
353 COL1A2 construct
containing sequences between
353 and +58 linked to the CAT reporter
gene was described previously (10). The
353 COL1A2/LUC was
generated by recloning the
353 to +58 promoter fragment into pGL2
basic (Promega). The EBS promoter mutants were constructed by replacing
guanosine in the EBS core sequence with thymidine using the
353
COL1A2 promoter construct as a template with the use of the
QuikChange site-directed mutagenesis kit (Stratagene) according to the
manufacturer's specifications (Table I). EBS mutations used in
electrophoretic mobility-shift assays (EMSAs) contain EBS 1-4
mutations. The COL1A2 promoter AP-1 binding site mutation
was generated as described previously (28). Fli/W321R contains a single
amino acid mutation that abolishes DNA binding. Fli/DBD contains DNA
binding domain 267-384. pSG5Fli-1, pSG5Ets-1, and pSG5Ets-2 were
described previously (20). Gal4-Sp1 and Gal4-Sp3 were a generous gift
of G. Suske (Marburg University, Germany). The PG5luc vector was
purchased from Promega.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of stable transfectants
overexpressing Fli-1. A, Fli-1 protein levels in
independent dermal fibroblast clones carrying empty vector
(Neo) or Fli-1 cDNA. In the first line (c) an
in vitro translated (TNT) Fli-1 was used. The expression
level of Fli-1 was examined by Western blot with a polyclonal
anti-human Fli-1 antibody. B, growth curve of Fli-1 (clone
5) and control transfectants. C, Fli-1 overexpression
decreases collagen protein level. Newly synthesized collagenous
proteins in control (Neo) and Fli-1 stable transfectants
were measured in a [3H]proline incorporation assay.
Conditioned medium normalized for cell number was analyzed for
collagenous protein content via SDS-polyacrylamide gel electrophoresis
and fluorography. D, Fli-1 overexpression inhibits
COL1A2 mRNA expression. The RNA isolated from the
control (empty vector) and Fli-1 stable transfectants was analyzed
simultaneously by Northern blot. Blots were hybridized with probes for
COL1A2 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as the loading control. E, a summary of
quantitative analysis of the Northern blots from three different
clones. All values were corrected for the loading differences by
normalizing to the glyceraldehyde-3-phosphate dehydrogenase mRNA
intensity.
3.5-kilobase
COL1A2 construct, whereas the
353 deletion was inhibited
about 1.7-fold. The
264 deletion produced variable results, although
the
186 and
108 deletions were modestly stimulated by Fli-1 (Fig.
2C). Thus, the inhibitory effect of Fli-1 was mediated by at
least two separate COL1A2 promoter regions: one region
located between bp
3500 and bp
772 and the second region located
between bp
353 and bp
185. Because the proximal 353-bp region of
the COL1A2 promoter is well characterized, we focused on
this region to further delineate the molecular mechanism of the
COL1A2 repression by Fli-1. Inspection of the nucleotide sequence of this promoter region revealed the presence of five putative
Fli-1 response elements (termed EBSs 1-5) located at bp
317,
307,
284,
244, and
200 (Fig. 2D). Significantly, EBSs 1-3 are located within the previously identified 60-bp
DNase-1-protected region (
322 to
262) that included three Sp1
binding sites (30).
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Fig. 2.
The effect of Fli-1 overexpression on
the COL1A2 promoter activity and identification of the
COL1A2 promoter region mediating Fli-1
inhibition. A, expression of exogenous Fli-1 protein.
Dermal fibroblasts were transfected with indicated amounts of
Fli-1/Flag expression vector or empty vector (pSG5) (line 1). Protein
levels of Fli-1/Flag in transfected cells were analyzed by
immunoprecipitation with anti-Flag M2 monoclonal antibody (Sigma), and
Western blot of the precipitates was analyzed with anti-Fli-1
polyclonal antibody. Equivalent amounts of protein were used for the
immunoprecipitations. B, dose-dependent
inhibition of the 353COL1A2/CAT promoter by
Fli-1. The COL1A2 promoter construct (0.1 µg) was
co-transfected with increasing concentrations of Fli-1 expression
vector. To ensure an equal amount of co-transfected expression vectors
under each condition, appropriate amounts of pSG5 were added to
individual co-transfections. C, transient transfections of
human dermal fibroblasts with the indicated deletion constructs of the
COL1A2/CAT promoter and either empty vector
(pSG5) or Fli-1 expression vector. The bar graph represents
the -fold induction of the promoter activity of each construct
co-transfected with Fli-1 vector relative to the activity of the
promoter co-transfected with empty vector (pSG5), which was arbitrarily
set at 1. The average of at least four independent experiments is
shown. The Fli-1-responsive element was mapped between
353 and
186.
(*, p
0.05). D, the nucleotide sequence
of the COL1A2 promoter region between bps
353 and
186.
The five potential Ets binding sites are boxed. Sp1/Sp3
binding sites are underlined.
353
COL1A2/CAT constructs carrying mutated GGAA
motifs were tested in transient transfection assays. Substitution mutation in the EBS 3 significantly reduced Fli-1 inhibition of the
COL1A2 promoter, whereas the mutations in other EBSs had no effect. (Fig. 3A). Because Ets
factors are known to cooperate with AP-1 in the regulation of urokinase
plasminogen activator and several MMP promoters (31), we asked whether
the previously characterized AP-1 site (28) in the COL1A2
promoter could be involved in the Fli-1 inhibition. The inhibitory
effect of Fli-1 on the COL1A2 promoter was not affected by
mutation of the previously characterized AP-1 response element.
However, the COL1A2 promoter containing mutations in the
three Sp1/Sp3 binding sites surrounding EBS 3 (see Fig. 2D)
become significantly less responsive to the inhibitory effects of Fli-1
(Fig. 3B). This is unexpected because Sp1 and Sp3 have been
shown to be potent activators of this promoter and their absence should
have resulted in a further decrease of the promoter activity. In fact,
promoter mutations in either Sp1/Sp3 or AP-1 binding sites result in a
decrease of the basal promoter activity; however, only mutations in the
Sp1/Sp3 binding sites affect the inhibitory response to Fli-1. This
unexpected result suggests that Sp1 and Sp3, in this promoter context,
may be considered co-repressors for Fli-1.
List of the EBS mutants in the COLIA2 promoter
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Fig. 3.
A, identification of a functional Fli-1
response element in the human COL1A2 promoter. The 353
COL1A2/CAT constructs harboring mutated EBSs
(EBSM) (see Fig. 2D, Table I, and "Materials
and Methods") were co-transfected with either Fli-1 expression vector
or empty vector (pSG5). The bar graph represents the -fold
induction of the promoter activity of each construct co-transfected
with Fli-1 relative to the activity of the promoter co-transfected with
pSG5, which was arbitrarily set at 1. Means ± S.E. of five
independent experiments are shown. Comparisons of COL1A2
promoter activity were made between cells transfected with empty vector
and with Fli-1 expression vector (*, < 0.01), and between cells
transfected with wild-type COL1A2 and EBS mutated
COL1A2 constructs (¶, < 0.01). B, Sp1/Sp3
binding is required for the inhibitory effect of Fli-1. The
COL1A2/CAT promoter constructs harboring either
Sp1/Sp3 triple mutations (Sp1TM) (30) or the AP-1 mutation
(AP1M) (28) were co-transfected with Fli-1 expression vector
or empty vector pSG5. Comparisons of COL1A2 promoter
activity were made between cells transfected with wild-type and mutated
promoter constructs (*, p < 0.01). The Fli-1
inhibitory effect on COL1A2 was diminished in Sp1/Sp3 triple
mutations.
307 to
269-bp COL1A2 promoter
fragment (39-bp oligomer) carrying all three Sp1 binding sites and
either wild-type or mutated EBS 3. EMSAs were performed using nuclear
extracts from human fibroblasts and a radiolabeled 39-mer probe (Fig.
4A). In agreement with
previously published data (9, 30), several specific DNA-protein
complexes were formed that could be competed by an excess of unlabeled
wild-type probe (lane 2). One of the complexes was not
competed by an excess of probe harboring EBS 3 mutation (lane
3), suggesting that the binding protein(s) in this complex is
related to Ets factor(s). Furthermore, formation of the same complex
was diminished when 39-mer-containing mutated EBS 3 was used as a probe
(lane 4). The presence of Sp1 and Sp3 in the complexes
formed with the wild-type and mutated probes were directly analyzed by
Western blotting with Sp1 and Sp3 antibodies. As shown in Fig.
4B, the EBS 3 mutation did not affect either Sp1 or Sp3
binding, suggesting that competition for DNA binding does not
contribute to the inhibitory effect of Fli-1.
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Fig. 4.
Sp1/Sp3 binding to the COL1A2
promoter is independent of EBS 3. A, EMSA with
competitor oligonucleotides. The wild-type probe (WT)
(lanes 1 to 3) or the EBS 3-mutated probe
(EBS3M) (lane 4) was incubated with human
fibroblast nuclear extracts (5 µg/lane) in the presence of wild-type
unlabeled competitor (50-fold molar excess) (lane 2) or EBS
3-mutated unlabeled competitor (lane 3). B,
detection of Sp1 and Sp3 in DNA-protein complexes. EMSAs were performed
with equal amounts of unlabeled wild-type and EBS 3-mutated probes,
followed by Western blot detection with anti-Sp1 and anti-Sp3
antibodies.
348 to
234-bp fragment of the
COL1A2 promoter and nuclear extracts from human dermal
fibroblasts. This promoter fragment was shown in our previous
footprinting analyses to contain a large (over 60-bp) protected area,
indicative of a multiprotein complex interacting with this promoter
region. Sp1 and Sp3 have been identified as the components of this
complex (9). Using EMSA we confirmed interaction of Sp1 and Sp3 with
the
348 to
234-bp promoter region (Fig.
5A, lane 3). Fli-1
also binds to this promoter region as indicated by a supershift with
the anti-Fli-1 antibody (lane 4). The ability of the
anti-Fli-1 antibody to shift Sp1 and Sp3 complexes in addition to the
Fli-1 complex suggests the occurrence of protein-protein interaction
between these proteins. To assess the effects of mutations in EBS on
binding of Fli-1, we used the
348 to
234 fragment containing
mutations in EBSs 1-4. Although functional assays indicated that EBS 3 mediates the Fli-1 response (Fig. 3), we chose to mutate other putative EBSs to test the possibility of binding to weaker potential sites. Neither Fli-1 nor Sp1 binding to this promoter region was affected by
EBS 1-4 mutations (Fig. 5B, compare lanes 1 and
3). Quantitation of the Fli-1 supershift band from nine
independent experiments shows no significant difference between Fli-1
binding to the wild-type probe and the probe containing EBS
mutations.
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Fig. 5.
Fli-1 interacts with the human
COL1A2 promoter. A, an EMSA with
antibodies against Sp1, Sp3, and Fli-1 was performed. The wild-type
fragment of the COL1A2 promoter ( 348 to
234)
(lanes 1-4) was incubated with nuclear extracts
(7 µg/lane) from human dermal fibroblasts in the presence of 1 µg
of specific antibody as indicated on the top. In lane
2, anti-human polyvalent IgG was used as a control.
Lane 1 contains probe alone. Specific DNA-protein
complexes and supershifted complexes are indicated by
arrows. B, Sp1/Sp3 binding facilitates Fli-1
binding to the human COL1A2 promoter. An EMSA was performed
with the wild-type probe (WT) (lanes
1-2), the probe containing mutated EBS 1-4
(EBSQM) (lanes 3-4), and the probe
containing mutated Sp1/Sp3 binding sites (Sp1TM)
(lanes 5-6). The probes were incubated with 7 µg of nuclear extract from dermal fibroblasts and 1 µg of specific
antibodies. Fli-1 binding was significantly decreased with the probe
containing mutated Sp1/Sp3 binding sites as indicated by diminished
supershift with anti-Fli-1 antibody. C, Ets-1 interacts with
the human COL1A2 promoter. The wild-type probe of the
COL1A2 promoter (
348 to
234) (lanes
1-4) was incubated with nuclear extracts (7 µg/lane)
from human fibroblasts in the presence of 1 µg of specific antibody
as indicated on the top. The arrow indicates the
Ets-1 supershift. Ab, antibody.
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Fig. 6.
Fli-1 mutants inhibit the COL1A2
promoter activity. Transient transfections were performed
with Fli-1 DNA binding mutant (Fli/W321R), Fli-1 dominant interference
mutant (Fli/DBD), and the 353 COL1A2 promoter. The Fli-1
DNA binding mutant contains a single amino acid mutation that abolishes
DNA binding and the Fli-1 dominant interference mutant contains the Ets
domain. Both Fli-1 mutants significantly decreased the
COL1A2 promoter activity (*, p
0.05) but
were less efficient than wild-type Fli-1.
and -
are expressed in dermal fibroblasts and that GABP
and -
are activators of the tenascin-C promoter in fibroblasts (20),
it is unlikely that GABP is involved in the COL1A2
regulation. GABP
and -
, which binds to DNA as a heterotetramer,
requires two tandem repeats of the GGA motif, which are not present in
the proximal region of the COL1A2 gene.
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Fig. 7.
Ets family members expressed in human
fibroblasts have distinct effects on the transcriptional activation of
the COL1A2 promoter. A, dermal
fibroblasts express Fli-1, Ets-1, and Ets-2. Lane 1,
corresponding control Ets TNT product; lane 2, endogenous
Ets protein. B, overexpression of Ets-1 stimulates the
COL1A2 promoter activity and abolishes Fli-1 inhibition of
the COL1A2 promoter in dermal fibroblast. Human dermal
fibroblasts were transiently co-transfected with the 353
COL1A2/LUC promoter construct (0.9 µg) and
either Fli-1 (0.1 µg) or Ets-1 (0.1 µg) was added individually or
with a constant amount of Fli-1 expression vector (0.1 µg) added
together with increasing amounts of Ets-1 (0.025, 0.05, and 0.1 µg)
expression vector. To ensure an equal amount of co-transfected
expression vectors under each condition, appropriate amounts of pSG5
were added to individual co-transfections. The bar graph
represents the -fold induction of the
353 COL1A2 promoter
activity co-transfected with Fli-1 or Ets-1 individually or together
relative to the activity of the promoter co-transfected with pSG5,
which was arbitrarily set at 1. The average ± S.E. from four
independent experiments is shown.
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Fig. 8.
A, Fli-1 and Ets-1 cooperate with Sp1
and Sp3 in activation of the Gal4 promoter in dermal fibroblasts. Human
dermal fibroblasts were co-transfected with 0.4 µg of a
Gal4-dependent luciferase (Gal4/LUC) reporter construct and
with 0.4 µg of expression constructs of fusion proteins containing
the DNA binding domain of Gal4 (Gal4DBD) and Sp1 and Sp3, Gal4-Sp1, and
Gal4-Sp3, respectively. In some experiments 0.1 µg of Fli-1 or Ets-1
expression vectors were also co-transfected. The bar graphs
represent means ± S.E. of the -fold induction of Gal4 promoter
activity from experimental conditions relative to the activity of the
Gal4 promoter co-transfected with Gal4DBD only, which was arbitrarily
set at 1. B, Ets-1 but not Fli-1 cooperates with Sp1 and Sp3
in activation of the Gal4 promoter in HepG2 cells. The same
experimental conditions were used in transfections with HepG2
cells.
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Fig. 9.
Transactivation of the COL1A2
promoter by Ets factors is cell
context-dependent. A, human lung
fibroblasts and mesangial , HepG2, and HeLa cells were transiently
co-transfected with the 353 COL1A2/LUC promoter
construct and Fli-1 or Ets-1 expression vectors. The bar graph
represents the -fold induction of the COL1A2 promoter
activity relative to the activity of the promoter co-transfected with
pSG5, which was arbitrarily set at 1. The average ± S.E. of at
least three independent experiments is shown (*, p
0.05). B, expression of endogenous Fli-1 and Ets-1
proteins in different cell types. C, corresponding
Ets TNT product; FB, foreskin fibroblasts; LF,
lung fibroblasts; HMC, human mesangial cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
activation of this promoter (8),
suggesting a possible role of TGF-
-induced signaling in this
process.
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Fig. 10.
A hypothetical model for Sp1-mediated
transcriptional repression or activation. Details described under
"Discussion."
Studies using dermal fibroblasts indicate that Fli-1 can act as a repressor in the context of the collagen promoter, and it is a potent activator for the tenascin-C promoter (20). What determines this dual function of Fli-1 is currently not known. Only a subset of Ets factors has repressor activity (e.g. ERF, YAN, TEL, and NET), and most are generally felt to be activators of transcription. However, previous studies have demonstrated that other proteins can block their ability to activate transcription via interaction with Ets. For example, interaction of EAP1/Daxx with Ets-1 has been shown to down-regulate Ets-1-mediated activation of MMP1 and BCL2 genes (37). In another system, MafB, an AP-1 like protein, has been shown to interact with the Ets domain of Ets-1 and repress Ets-1 transactivation of Ets-responsive promoters (38). More importantly, Fli-1 has been shown to be an interacting partner of TEL, a member of the Ets family with repressor function. Furthermore, co-transfection of TEL with Fli-1 has been shown to inhibit Fli-1-mediated transcriptional activation (39). TEL contains two autonomous repression domains, suggesting two distinct repression mechanisms (40). One of the potential mechanisms involves the recruitment of a repression complex including silencing mediator for retinoid and thyroid hormone receptors and mSin3A (41). Whether TEL is involved in Fli-1-mediated repression of the COL1A2 remains to be elucidated. Significantly, however, stable overexpression of TEL in NIH3T3 fibroblasts led to the down-regulation of collagen type I (42).
In support of the conclusions from our studies, it was observed that
the exogenous expression of Ewing's sarcoma protein/FLI1 and Ewing's
sarcoma protein/ETV1 inhibited deposition of collagen in NIH3T3 cells
(43). Significantly, we have found that a subset of lesional
scleroderma fibroblast cell lines expresses reduced levels of Fli-1
protein as compared with healthy control fibroblasts, whereas the
expression of Ets-1 remains constant. The reduced levels of Fli-1
correlate with increased collagen production by these
cells.3 Thus, Fli-1 and other
Ets factors may play a role in the pathology of fibrotic diseases. It
will be important to examine in vivo expression of Ets
factors in fibrotic lesions and in animal models of fibrosis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Suske for plasmids, Dr. Tien Hsu for critical reading of the manuscript, and Martin Trojanowski for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AR 42334, NCI National Institutes of Health Grant PO1 CA78582, and the R. G. Kozmetsky Foundation.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.
§ Both authors contributed equally to this work.
¶ Recipient of a grant from the Zarkin Fund of Boston.
Division of Rheumatology and Immunology, Medical University of
South Carolina, 96 Jonathan Lucas St., Ste. 912, Charleston, SC 29401. Tel.: 843-792-7921; Fax: 843-792-7121; E-mail:
trojanme@musc.edu.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M010133200
2 D. K. Watson, unpublished observations.
3 J. Czuwara-Ladykowska and M. Trojanowska, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
COL1A2, type I collagen 2(I);
TGF, transforming
growth factor;
MMP, matrix metalloproteinase;
EBS, Ets binding site;
EMSA, electrophoretic mobility-shift assay;
DBD, DNA binding domain;
bp, base pair;
GABP, GA-binding protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Vincenti, M. P., Clark, I. M., and Brinckerhoff, C. E. (1994) Arthritis Rheum. 37, 1115-1126[Medline] [Order article via Infotrieve] |
2. | Malemud, C. J., and Goldberg, V. M. (1999) Front. Biosci. 4, D762-D771[Medline] [Order article via Infotrieve] |
3. | Forget, M. A., Desrosiers, R. R., and Beliveau, R. (1999) Can. J. Physiol. Pharmacol. 77, 465-480[CrossRef][Medline] [Order article via Infotrieve] |
4. | Curran, S., and Murray, G. I. (1999) J. Pathol. 189, 300-308[CrossRef][Medline] [Order article via Infotrieve] |
5. | Trojanowska, M., LeRoy, E. C., Eckes, B., and Krieg, T. (1998) J. Mol. Med. 76, 266-274[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Friedman, S. L.
(1993)
N. Engl. J. Med.
328,
1828-1835 |
7. | Border, W. A., Yamamoto, T., and Noble, N. A. (1996) Diabetes Metab. Rev. 12, 309-339[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Zhang, W.,
Ou, J.,
Inagaki, Y.,
Greenwel, P.,
and Ramirez, F.
(2000)
J. Biol. Chem.
275,
39237-39245 |
9. |
Ihn, H.,
and Trojanowska, M.
(1997)
Nucleic Acids Res.
25,
3712-3717 |
10. |
Ihn, H.,
Ohnishi, K.,
Tamaki, T.,
LeRoy, E. C.,
and Trojanowska, M.
(1996)
J. Biol. Chem.
271,
26717-26723 |
11. | Ghosh, A. K., Yuan, W., Mori, Y., and Varga, J. (2000) Oncogene 19, 3546-3555[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Chen, S. J.,
Yuan, W.,
Mori, Y.,
Levenson, A.,
Trojanowska, M.,
and Varga, J.
(1999)
J. Invest. Dermatol.
112,
49-57 |
13. | Chen, S. J., Yuan, W., Lo, S., Trojanowska, M., and Varga, J. (2000) J. Cell. Physiol. 183, 381-392[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Kouba, D. J.,
Chung, K. Y.,
Nishiyama, T.,
Vindevoghel, L.,
Kon, A.,
Klement, J. F.,
Uitto, J.,
and Mauviel, A.
(1999)
J. Immunol.
162,
4226-4234 |
15. |
Greenwel, P.,
Tanaka, S.,
Penkov, D.,
Zhang, W.,
Olive, M.,
Moll, J.,
Vinson, C.,
Di Liberto, M.,
and Ramirez, F.
(2000)
Mol. Cell. Biol.
20,
912-918 |
16. |
Poncelet, A. C.,
and Schnaper, H. W.
(2000)
J. Biol. Chem.
276,
6983-6992 |
17. | Graves, B. J., and Petersen, J. M. (1998) Adv. Cancer Res. 75, 1-55[Medline] [Order article via Infotrieve] |
18. |
Spyropoulos, D. D.,
Pharr, P. N.,
Lavenburg, K. R.,
Jackers, P.,
Papas, T. S.,
Ogawa, M.,
and Watson, D. K.
(2000)
Mol. Cell. Biol.
20,
5643-5652 |
19. | Mager, A. M., Grapin-Botton, A., Ladjali, K., Meyer, D., Wolff, C. M., Stiegler, P., Bonnin, M. A., and Remy, P. (1998) Int. J. Dev. Biol. 42, 561-572[Medline] [Order article via Infotrieve] |
20. | Shirasaki, F., Makhluf, H. A., LeRoy, C., Watson, D. K., and Trojanowska, M. (1999) Oncogene 18, 7755-7764[CrossRef][Medline] [Order article via Infotrieve] |
21. | Calmels, T. P., Mattot, V., Wernert, N., Vandenbunder, B., and Stehelin, D. (1995) Biol. Cell 84, 53-61[CrossRef][Medline] [Order article via Infotrieve] |
22. | Bolon, I., Gouyer, V., Devouassoux, M., Vandenbunder, B., Wernert, N., Moro, D., Brambilla, C., and Brambilla, E. (1995) Am. J. Pathol. 147, 1298-1310[Abstract] |
23. | Wasylyk, C., Gutman, A., Nicholson, R., and Wasylyk, B. (1991) EMBO J. 10, 1127-1134[Abstract] |
24. | Wasylyk, B., Hagman, J., and Gutierrez-Hartmann, A. (1998) Trends Biochem. Sci. 23, 213-216[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Cirillo, G.,
Casalino, L.,
Vallone, D.,
Caracciolo, A.,
De Cesare, D.,
and Verde, P.
(1999)
Mol. Cell. Biol.
19,
6240-6252 |
26. | Trojanowska, M. (2000) Oncogene 19, 6464-6471[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ichiki, Y., Smith, E. A., LeRoy, E. C., and Trojanowska, M. (1997) J. Rheumatol. 24, 90-95[Medline] [Order article via Infotrieve] |
28. |
Chung, K. Y.,
Agarwal, A.,
Uitto, J.,
and Mauviel, A.
(1996)
J. Biol. Chem.
271,
3272-3278 |
29. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
30. |
Tamaki, T.,
Ohnishi, K.,
Hartl, C.,
LeRoy, E. C.,
and Trojanowska, M.
(1995)
J. Biol. Chem.
270,
4299-4304 |
31. | Crawford, H. C., and Matrisian, L. M. (1996) Enzyme Protein 49, 20-37[Medline] [Order article via Infotrieve] |
32. | Bailly, R. A., Bosselut, R., Zucman, J., Cormier, F., Delattre, O., Roussel, M., Thomas, G., and Ghysdael, J. (1994) Mol. Cell. Biol. 14, 3230-3241[Abstract] |
33. |
Dittmer, J.,
Pise-Masison, C. A.,
Clemens, K. E.,
Choi, K. S.,
and Brady, J. N.
(1997)
J. Biol. Chem.
272,
4953-4958 |
34. |
Doetzlhofer, A.,
Rotheneder, H.,
Lagger, G.,
Koranda, M.,
Kurtev, V.,
Brosch, G.,
Wintersberger, E.,
and Seiser, C.
(1999)
Mol. Cell. Biol.
19,
5504-5511 |
35. |
Murphy, M.,
Ahn, J.,
Walker, K. K.,
Hoffman, W. H.,
Evans, R. M.,
Levine, A. J.,
and George, D. L.
(1999)
Genes Dev.
13,
2490-2501 |
36. | Gu, W., and Roeder, R. G. (1997) Cell 90, 595-606[Medline] [Order article via Infotrieve] |
37. | Li, R., Pei, H., Watson, D. K., and Papas, T. S. (2000) Oncogene 19, 745-753[CrossRef][Medline] [Order article via Infotrieve] |
38. | Sieweke, M. H., Tekotte, H., Frampton, J., and Graf, T. (1996) Cell 85, 49-60[Medline] [Order article via Infotrieve] |
39. |
Kwiatkowski, B. A.,
Bastian, L. S.,
Bauer, T. R.,
Tsai, S.,
Zielinska- Kwiatkowska, A. G.,
and Hickstein, D. D.
(1998)
J. Biol. Chem.
273,
17525-17530 |
40. |
Lopez, R. G.,
Carron, C.,
Oury, C.,
Gardellin, P.,
Bernard, O.,
and Ghysdael, J.
(1999)
J. Biol. Chem.
274,
30132-30138 |
41. | Chakrabarti, S. R., and Nucifora, G. (1999) Biochem. Biophys. Res. Commun. 264, 871-877[CrossRef][Medline] [Order article via Infotrieve] |
42. | Van Rompaey, L., Dou, W., Buijs, A., and Grosveld, G. (1999) Neoplasia 1, 526-536[CrossRef][Medline] [Order article via Infotrieve] |
43. | Teitell, M. A., Thompson, A. D., Sorensen, P. H., Shimada, H., Triche, T. J., and Denny, C. T. (1999) Lab. Invest. 79, 1535-1543[Medline] [Order article via Infotrieve] |