From the Section of Rheumatology, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607
Received for publication, May 31, 2000, and in revised form, December 28, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Among the extracellular signals that modulate the
synthesis of collagen, transforming growth factor- Transforming growth factor- TGF- We have shown previously that overexpression of Smad3 in primary skin
fibroblasts mimicked the action of TGF- CREB-binding protein (CBP) and adenovirus E1A-associated protein p300
are structurally conserved large proteins that function as essential
coactivators in several signal transduction pathways. p300/CBP enhance
transcription by bridging DNA-bound factors and the basal
transcriptional machinery, thereby stabilizing the pre-initiation complex. Furthermore, by nature of their intrinsic histone
acetyltransferase enzyme activity, these coactivators acetylate
amino-terminal lysine residues of nucleosomal histones (27, 28). Local
histone hyperacetylation causes nucleosomal relaxation, thus promoting
access of transcription factors to target DNA sequences. The
transcriptional activity of genes is strongly correlated with their
acetylation state (29). The presence of conserved protein-binding
domains enables p300/CBP to interact with distinct classes of regulated
transcription factors. Through binding to CREB, the p65 component of
NF- Interferon- As well as suppressing the stimulation of collagen transcription,
IFN- Reagents and Cell Culture--
All tissue culture reagents were
obtained from Life Technologies, Inc. (Grand Island, NY). Recombinant
human IFN- Immunoprecipitation and Western Immunoblotting--
At the end
of the indicated period of incubation, cells were washed with ice-cold
phosphate-buffered saline, and whole cell lysates prepared. Lysates
were either directly resolved by electrophoresis in polyacrylamide
gels, or first immunoprecipitated for 1 h using anti-p300 antibody
(Santa Cruz Biotechnology, Santa Cruz CA). Gels were blotted onto
polyvinylidene difluoride membranes (Millipore, Bedford, MA), and
subjected to immunoblotting with primary antibodies (human anti-p300,
anti-Smad7, and anti-actin (C-2) from Santa Cruz; anti-Smad3 from
Zymed Laboratories Inc., San Francisco, CA; anti-Type
I collagen from Southern Biotech (Birmingham, AL); anti-Stat1 from
Transduction Labs (Lexington, KY); and anti-phosphotyrosine Stat1
(Y701) from Upstate Biotechnology (Lake Placid, NY) for 2 h at
room temperature. The blots were then washed, followed by incubation
with the appropriate horseradish peroxidase-conjugated secondary
antibody, and visualized by chemiluminescence.
Cellular Immunofluorescence Imaging--
The expression and
intracellular localization of endogenous SMADs and Stat1 Transient Transfection--
To measure transcriptional responses
to IFN- Preparation of Nuclear Extracts and Electrophoretic Gel Mobility
Shift Assay--
Fresh media with 0.2% fetal calf serum and IFN- Statistical Analysis--
Statistical differences between
experimental groups were determined by analysis of variance, and values
of p < 0.05 by Fisher's test were considered significant.
Antagonistic Regulation of Collagen Transcription by TGF-
Because we have previously established a fundamental role for Smad3 in
TGF- Smad7-independent Inhibition of COL1A2 Transcription in
Fibroblasts--
Intracellular cross-talk among cytokines with
opposing effects can be mediated through induction by one cytokine of
autocrine mediators that block signaling triggered by the another
cytokine. For instance, IFN-
We next examined the functional consequences of down-regulating
endogenous Smad7 using an antisense expression plasmid (59). Fibroblasts were transfected with 10 µg of 772COL1A2/CAT
along with 5 µg of antisense Smad7 cDNA, or empty
vector. As expected, transactivation of COL1A2 by TGF- IFN-
IFN- TGF-
To directly examine the in vivo interaction of endogenous
Smad3 with p300, fibroblasts were transfected with a p300
expression plasmid and treated with TGF- The production of ECM proteins by fibroblasts must be coordinated
and strictly regulated. During dynamic processes of tissue remodeling
such as wound healing, fibroblasts are subject to simultaneous signaling by distinct combinations of cytokines, growth factors, and
other regulatory molecules. These extracellular signals provide fibroblasts with information that synergistically or antagonistically influence the expression of target genes. Type I collagen is the major
structural component of the connective tissue of the skin and other
organs. Because its excessive accumulation results in fibrosis, the
dynamic equilibrium between signals that stimulate and those that
inhibit collagen synthesis must be carefully maintained. The
transcription of COL1A2, one of the best characterized
responses in fibroblasts, is stimulated by TGF- The results presented here provide evidence for the nuclear integration
of TGF- (TGF-
) and
interferon-
(IFN-
) are preeminent. These two cytokines exert
antagonistic effects on fibroblasts, and play important roles in the
physiologic regulation of extracellular matrix turnover. We have shown
previously that in normal skin fibroblasts, TGF-
positively
regulates
2(I) procollagen gene (COL1A2) promoter
activity through the cellular Smad signal transduction pathway. In
contrast, IFN-
activates Stat1
, down-regulates COL1A2
transcription, and abrogates its stimulation induced by TGF-
. The
level of integration of the two pathways mediating antagonistic
collagen regulation is unknown. We now report that IFN-
abrogates
TGF-
-stimulated COL1A2 transcription in fibroblasts by
inhibiting Smad activities. IFN-
appears to induce competition
between activated Stat1
and Smad3 for interaction with limiting
amounts of cellular p300/CBP. Overexpression of p300 restored
COL1A2 stimulation by TGF-
in the presence of IFN-
, and potentiated IFN-
-dependent positive transcriptional
responses. In contrast to fibroblasts, in U4A cells lacking Jak1 and
consequently unable to activate Stat1
-mediated responses, IFN-
failed to repress TGF-
-induced transcription. These results indicate
that as essential coactivators for both Smad3 and Stat1
, nuclear
p300/CBP integrate signals that positively or negatively regulate
COL1A2 transcription. The findings implicate a novel
mechanism to account for antagonistic interaction of Smad and Jak-Stat
pathways in regulation of target genes. In fibroblasts responding to
cytokines with opposing effects on collagen transcription, the relative levels of cellular coactivators, and their interaction with regulated transcription factors, may govern the net effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
regulates cellular responses
through modulation of transcription of genes encoding cell cycle regulators, extracellular matrix proteins, adhesion molecules, cytokines, and transcription factors (1). One of the most potent effects of TGF-
1 is
connective tissue accumulation, achieved in part by stimulation of the
transcription of type I collagen genes (2-5). Thus, TGF-
plays
crucial roles in embryonic development and organogenesis, and
physiologic connective tissue remodeling during wound healing and
tissue repair. On the other hand, excessive TGF-
activity is
implicated in the development of pathological fibrosis, the "dark
horse of tissue repair" (6).
initiates cellular signals through two distinct transmembrane
serine-threonine kinase receptors. Upon ligand binding, the activated
TGF-
receptor complex transiently interacts with receptor-activated
Smads which propagate TGF-
signals (7). Smad2 and Smad3 are direct
substrates of the TGF-
receptor kinase, and interact with the common
partner Smad4 (8-10). Smad4-containing heteromeric Smad complexes then
translocate from the cytoplasm into the nucleus where they function as
transcriptional regulators. Smads2-4 share highly conserved
DNA-binding MH1 and transactivating MH2 domains; the latter also
mediates Smad interactions with other proteins (11, 12). Smad7 contains
a characteristic MH2 domain, but lacks the conserved SSXS
phosphorylation motif, and its MH1 domain shows marked divergence from
that of Smad3 (13). Furthermore, in contrast to receptor-activated
Smads, Smad7 stably binds to TGF-
receptors and interferes with
ligand-induced phosphorylation of Smad2 and Smad3 (14). Because its
expression is markedly induced by the ligand, Smad7 appears to serve an
autoregulatory negative feedback function in cellular TGF-
signaling.
, markedly inducing COL1A2 promoter activity in vitro (15).
Transactivation was blocked by phosphorylation-defective dominant
negative mutants of Smad3, establishing the critical role of endogenous
Smads in transducing information from the activated TGF-
receptor in
these cells. The COL1A2 promoter contains
Smad3/Smad4-binding consensus "CAGA boxes" (16), also found in the
promoters of PAI-1, junB, and other TGF-
inducible genes (17, 18), that are necessary and sufficient to mediate
transcriptional responses induced by TGF-
(19). However, as
CAGA boxes are widely distributed in the promoters of
mammalian genes, and the affinity and specificity of SMAD binding to
these is relatively low, other nuclear factors are likely to contribute
to the specific and tight Smad-DNA interactions that are required for
transcriptional regulation (20). Cooperation with FAST-1 (21), AP-1
(22, 23), TFE3 (24), Sp1 (25), and vitamin D receptor (26) are
implicated in Smad-mediated transcription of TGF-
-responsive genes.
B, the c-Jun, and c-Fos components of AP-1, and Stats, p300/CBP
integrate converging cellular signaling pathways (30-35).
Receptor-activated Smads interact directly with p300/CBP via the Smad
MH2 domain and the p300 COOH-terminal region overlapping the
E1A-binding site (36-41). We previously demonstrated that p300
markedly enhanced TGF-
-stimulated COL1A2 transcription in
fibroblasts (42). By competing with activated Smad3 for limiting
amounts of cellular p300/CBP, the adenoviral oncoprotein E1A abrogated
TGF-
transactivation, indicating the critical role of p300/CBP in
collagen gene transcription in fibroblasts.
(IFN-
), a pleiotropic cytokine produced by T cells
and NK cells, plays fundamental roles in both innate and acquired
immune responses (43). Transcriptional responses induced by IFN-
in
most cells are mediated through the Jak-Stat pathway (44). Upon
stimulation by IFN-
, tyrosine-phosphorylated cytoplasmic Stat1
forms homodimeric complexes that can translocate into the nucleus, and
bind directly to palindromic
-activated sites (GAS) of
IFN-
-responsive target gene promoters. Stat1
thus serves as an
essential mediator of IFN-
-induced transcriptional responses. Stat1
physically associates with p300/CBP near its amino-terminal domain; this interaction plays an important functional role in positive
regulation of IFN-
-induced transcriptional responses (45, 46). In
addition to transcriptional stimulation, IFN-
can also negatively
regulate the transcription of selected genes, but no common
IFN-
-specific inhibitory elements have been identified. We and
others have shown previously that IFN-
inhibits the transcription of
collagen in fibroblasts independent of Stat1-promoter interactions, and
abrogates its stimulation induced by TGF-
(47-51). Thus, TGF-
and IFN-
exert opposite effects on collagen synthesis. Because these
two cytokines are secreted by inflammatory cells at sites of tissue
injury, their antagonistic interactions regulating collagen synthesis
are likely to be of great importance in the maintenance of connective
tissue homeostasis.
abrogates other TGF
responses, including collagenase-3 expression in epithelial cells (52), perlecan expression in colon
carcinoma cells (53), and fibronectin and laminin receptor expression
in monocytic cells (54). The basis underlying antagonistic modulation
of TGF-
signaling by IFN-
is incompletely understood. In the
present report, we characterized the repression of TGF-
-stimulated collagen transcription by IFN-
in normal skin fibroblasts. The results indicate that the stimulatory effects of TGF-
on Type I
collagen gene (COL1A2) transcription were abrogated by
IFN-
through a minimal Smad-binding element of the COL1A2
promoter. Inhibition of TGF-
signaling in fibroblasts was not
mediated through antagonistic Smad7. The stimulatory effect of TGF-
on COL1A2 transcription could be rescued in the presence of
IFN-
by overexpression of p300. These findings indicate that
p300/CBP integrate IFN-
/TGF-
-induced signals that positively or
negatively regulate collagen gene transcription in fibroblasts, and
suggest that an increase in activated Stat1
in IFN-
-treated
fibroblasts suppressed Smad-mediated transactivation by titrating away
the coactivators. The findings provide novel understanding of the physiologically important antagonistic regulation of collagen gene
transcription by cytokines.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was from Genentech Inc. (South San Francisco, CA), and
TGF-
from Amgen (Thousand Oaks, CA). Primary cell cultures were
established from neonatal foreskin by previously described explant
techniques (15), and studied between passages 4 and 8. Cells were grown
at 37 °C in a 5% CO2 atmosphere in modified Eagle's
medium supplemented with 1% or 10% fetal calf serum, 1%
vitamins, and 2 mM L-glutamine. The U4A
Jak1-deficient cell line, which does not support
IFN-
-induced gene expression (55), and U4A/Jak1 were a
kind gift of O. Colamonici. Transforming growth factor-
and IFN-
were added simultaneously, and cultures were harvested following 24-48
h incubation. In previous studies under similar conditions, we found
that neither TGF-
nor IFN-
significantly effected cell number or
viability in confluent fibroblast cultures (15). Total RNA was isolated
with TRIZOL Reagent (Life Technologies, Inc.) following the indicated
periods of incubation, and relative levels of mRNA were examined by
Northern analysis using radiolabeled Smad3, Smad7, and
glyceraldehyde-3-phosphate dehydrogenase cDNA probes.
The cDNA-mRNA hybrids were visualized by autoradiography on
Kodak X-AR5 films exposed for 24-48 h with intensifying screens.
in the
presence or absence of IFN-
or TGF-
was studied by indirect
immunofluorescence. For this purpose, fibroblasts were seeded into
chamber glass slides and incubated in media with 0.1% fetal calf serum
and IFN-
added 30 min before TGF-
. After 2 h incubation,
cells were then fixed with methanol, incubated with primary antibodies
(anti-Smad3 and anti-Smad4 from Santa Cruz; anti-Stat1
from
Transduction Labs) for 1 h, followed by horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz). After three
washes, the slides were stained, and the intracellular distribution of
Smads and Stat1 was examined by fluorescence or confocal microscopy.
Quantitation was performed in a blinded fashion by scoring 100 fibroblasts in different fields as showing predominantly nuclear or
cytoplasmic immunofluorescence.
or TGF-
, 772COL1A2/CAT consisting of the
772
to +58 bp segment of human COL1A2 promoter linked to the
CAT gene (56), CAGA-COL1A2/luc containing six
copies of the
266/
258 bp sequence of COL1A2 (17), or
SBE4-luc containing four tandem repeats of an 8-bp
palindromic consensus Smad-binding consensus sequence (16) were used.
Expression vectors for wild type and
HAT-mutant p300
(57), Smad3 (58), and antisense Smad7 cDNA
(59), and appropriate control vectors were transfected. As controls,
-152DR-CAT (from J. Ting, University of North Carolina, Chapel Hill, NC) containing an IFN-
-responsive 152-bp segment of the
HLA-DR promoter ligated to the CAT gene (60), and
GAS-tk-CAT (from G. Sen, Cleveland Clinic Foundation
Research Institute) containing an IFN-
-responsive 24-bp segment of
the IRF-1 promoter ligated to the CAT gene, were used.
Transient transfections were performed by calcium phosphate/DNA
co-precipitation or using Superfect reagent (Qiagen, Valencia, CA), as
described previously (15). The total amount of DNA in each transfection
was kept constant by addition of appropriate empty vectors, as
required. Following incubation with the indicated cytokines, CAT, and
luciferase activities were determined in duplicate and normalized with
protein concentration.
alone or with TGF-
were added to confluent fibroblasts. At the end
of the indicated incubation periods, nuclear extracts were prepared
according to the method of Andrews and Faller (61), and protein
concentrations were determined using the Bio-Rad assay. Double-stranded
oligonucleotide probes corresponding to a consensus Smad-binding
element (SBE). The probes were end-labeled using T4
polynucleotide kinase (Promega, Madison WI). Electrophoretic gel
mobility shift assays were performed as described previously (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
IFN-
, Convergence at the Level of Smad3--
To examine the
regulation of collagen transcription, normal skin fibroblasts were
transiently transfected with COL1A2 promoter-CAT reporter constructs. TGF-
caused marked stimulation of CAT activity driven by 772 bp of the COL1A2 promoter. This induction was
completely abrogated by IFN-
, indicating that cis-elements necessary
for negative regulation of COL1A2 expression were contained
within this region of the promoter (Fig.
1A, left panel). The activity of
152DR-CAT was induced by IFN-
, demonstrating that
the inhibitory effect of IFN-
was selective for the
COL1A2 promoter (Fig. 1A, middle panel). Changes
in the intracellular pool of collagen in response to the cytokines
paralleled those in promoter activity (Fig. 1B). Inhibition
of collagen synthesis did not require pretreatment with IFN-
prior
to stimulation. IFN-
induced the rapid nuclear translocation of
Stat1, an essential mediator of IFN-
transcriptional responses (Fig.
2). To further examine Stat1 activation,
the consensus Stat1
-binding GAS oligonucleotide was used
as probe in gel shifts. As expected, IFN-
treatment of fibroblasts
for 15 min resulted in the formation of a GAS-specific
DNA-protein complex (Ref. 62; and data not shown). A careful analysis
of the proximal COL1A2 promoter failed to identify the
presence of consensus Stat1
-binding GAS elements
(TTN5AA). Furthermore, in contrast to classical
Stat1-mediated transcriptional responses, the inhibitory effect of
IFN-
on COL1A2 promoter activity was delayed (>8 h)
(51). Therefore, these observations suggested that Stat1
activation
could not be fully responsible for suppression of COL1A2
transcription by IFN-
.
View larger version (22K):
[in a new window]
Fig. 1.
Stimulation of collagen transcription is
abrogated by IFN- . A,
fibroblasts were transfected with 772COL1A2/CAT (left
panel), the IFN-
responsive minimal promoter
-DR152-CAT (middle panel), or
CAGA-COL1A2/luc, which contains tandem copies of the COL1A2
Smad-binding consensus CAGA sequence (right panel).
Following 48 h incubation with IFN-
(500 units/ml) and/or
TGF-
(500 pM), cultures were harvested and CAT and luc
activities were determined. The results for 772COL1A2/CAT
and CAGA-COL1A2/luc, shown as relative reporter gene
activity, indicate the means of corrected duplicates from three
independent experiments. Schematic representation for the constructs
used is shown below. B, levels of Type I collagen in
fibroblasts treated with TGF-
and/or IFN-
for 48 h was
determined by Western blot of whole cell lysates. A representative
autoradiogram is shown on top. The intensities of the bands
quantiated by densitometry and corrected for actin are shown at the
bottom.
View larger version (51K):
[in a new window]
Fig. 2.
IFN- induces Stat1
activation in fibroblasts. Fibroblasts were treated with
IFN-
for 2 h, or left untreated. At the end of the incubation
period, cells were processed for immunocytochemistry, and
STAT1-specific immunofluorescence was detected by confocal microscopy,
as described under "Materials and Methods."
stimulation of COL1A2 transcription in fibroblasts (15), the possibility that the Smad signal transduction pathway was a
target for the inhibitory activities of IFN-
was considered. To
directly examine the involvement of Smad3, the heterologous minimal
construct CAGA-COL1A2/luc, which contains six
tandem copies of the COL1A2 Smad-binding CAGA
element shown to be sufficient to mediate TGF-
responses (19) was
used. The TGF-
-induced increase in activity of this promoter was
suppressed in IFN-
-treated fibroblasts (Fig. 1A, right
panel). Identical results were obtained with SBE4-luc,
containing a consensus SBE (16). IFN-
had no effect on
endogenous Smad3 or Smad4 mRNA or protein expression (data not
shown). Together, these findings indicated that IFN-
suppressed
TGF-
stimulation of COL1A2 transcription mediated by
multimerized Smad3-recognition sites, while inducing endogenous Stat1
activation. Inhibition did not result from decreased
expression or DNA binding of cellular Smads, suggesting instead that
IFN-
targeted their transcriptional activities.
abrogates interleukin-4-induced
transcriptional responses via endogenous suppressor of cytokine
signaling SOCS-1, which prevented STAT6 activation in these cells (63,
64). In a similar vein, TGF-
stimulation of 3TP-lux
transcription is prevented by IFN-
or TNF-
via induction of
antagonistic cellular Smad7, which blocks ligand-induced Smad3
phosphorylation and its attendant events through stable interaction
with the TGF-
receptors (65, 66). These observations suggested a
possible mechanism to account for the antagonistic effect of IFN-
on
TGF-
-stimulated COL1A2 transcription: induction of an
endogenous inhibitor of TGF-
signaling by IFN-
. In contrast to
SOCS-1, which has not been shown to suppress TGF-
signaling, Smad7
specifically blocked Smad3-mediated responses in fibroblasts (15).
Therefore, we sought to examine whether endogenous Smad7 could be
implicated in IFN-
suppression of TGF-
-stimulated
COL1A2 transcription. The results showed that, whereas it
consistently induced rapid and transient increase in Smad7 mRNA
expression in HepG2 epithelial cells, IFN-
had no detectable effect
on the levels of endogenous Smad7 determined by immunocytochemistry and
Western immunoblotting, or on Smad7 mRNA expression determined by
Northern analysis (data not shown). Our failure to demonstrate
induction of Smad7 expression by IFN-
in fibroblasts, in contrast to
cells of epithelial origin, is consistent with recent report (66).
was greater in fibroblasts expressing antisense Smad7 than
in control fibroblasts transfected with empty vector, indicating that
the level of endogenous Smad7 determined the magnitude of the
TGF-
-induced response (Fig.
3A). Smad7
antisense cDNA markedly decreased the amount of cellular Smad7 (Fig. 3B). The inhibitory effect of antisense
on cellular Smad7 was unaffected by IFN-
(data not shown).
Down-regulation of endogenous Smad7 did not abrogate the inhibitory
effect of IFN-
on TGF-
-induced COL1A2 promoter in
these fibroblasts (Fig. 3A). Similar results were obtained
when antisense oligonucleotides were used to down-regulate endogenous
Smad7 (data not shown). Taken together, these results indicate that
endogenous Smad7 is not responsible for abrogation of
TGF-
-stimulated COL1A2 transcription by IFN-
in
primary fibroblasts.
View larger version (17K):
[in a new window]
Fig. 3.
Antisense Smad7 does not
prevent inhibition of TGF- -induced
COL1A2 transcription by
IFN-
. A, fibroblasts were
co-transfected with 772COL1A2/CAT (10 µg) along with
antisense Smad7 expression constructs (5 µg) or empty
vector. Six h later, TGF-
(500 pM) was added to the
cultures. Following further 24 h incubation in media with 0.1%
fetal calf serum without or with IFN-
and/or TGF-
, cultures were
harvested and CAT activities were determined. The results, shown as
-fold change in CAT activity, are expressed as the means of duplicates
from three independent experiments. B, whole cell lysates
from fibroblasts transfected with pcDNA3 or
antisense Smad7 (AS-Smad7) were subjected to
immunoblotting with antibodies against Smad7 or actin.
Abrogates COL1A2 Stimulation Downstream from Smad3
Activation--
The antagonistic effect of IFN-
on Smad-mediated
COL1A2 transactivation could result from interference with
TGF-
-induced Smad3 activation. Three complementary approaches were
undertaken to examine this possibility. First, we sought to determine
whether inhibition of Smad-mediated transactivation by IFN-
altered
the DNA binding activity of nuclear proteins. Electrophoretic mobility shift assays with fibroblast nuclear extracts showed that incubation with IFN-
slightly enhanced TGF-
-induced binding of endogenous Smad3 to the consensus SBE (Fig.
4A). To assess the effects of IFN-
on the transactivation function of Smad3, fibroblasts
overexpressing recombinant Smad3 were treated with IFN-
. As shown in
Fig. 4B, the striking elevation in COL1A2
promoter activity in Smad3-transfected fibroblasts was
substantially abrogated, indicating that in these cells IFN-
disrupted signaling downstream of the activated TGF-
receptor.
IFN-
did not alter the level of recombinant Smad3 expression (Fig.
4B, inset; and data not shown). Next, the effect of IFN-
on the subcellular distribution of endogenous Smad3 was examined. Nuclear import of Smad3, essential for regulating target gene transcription, is highly dependent on its ligand-induced
phosphorylation (11). Treatment of the fibroblasts with TGF-
caused
rapid (<2 h) accumulation of endogenous Smad3 and Smad4 within the
nucleus (Fig. 4C). IFN-
by itself had no effect on Smad
cellular localization, and pretreatment of the cells failed to prevent
TGF-
-induced nuclear translocation at early or late time points. The
inability of IFN-
to disrupt ligand-induced nuclear import of
activated Smads further indicates that mechanisms distinct from
blocking ligand-induced Smad activation underlie the antagonistic
effects of IFN-
on TGF-
stimulation of COL1A2
transcription.
View larger version (39K):
[in a new window]
Fig. 4.
Antagonistic regulation of Smad-mediated
transcriptional responses by IFN- .
A, confluent fibroblasts were incubated with TGF-
and/or
IFN-
for 60 min. Nuclear extracts were analyzed in gel mobility
shift assays with a radiolabeled oligonucleotide probe containing the
SBE core sequence. The shifted band identified as Smad is
indicated by an arrow. B, fibroblasts were co-transfected
with a plasmid expressing Smad3 along with 772COL1A2/CAT.
Following 48 h incubation of the cultures in media without
(open bars) or with IFN-
(500 units/ml, closed
bars), CAT activities were determined. The results shown represent
the mean of three independent determinations; *, p < 0.05. Inset, expression of recombinant Smad3 unaffected in
fibroblasts treated with IFN-
. Equal amounts of whole cell lysates
from untreated (lanes 1 and 3) or IFN-
-treated
(lanes 2 and 4) fibroblasts transiently
transfected with plasmid expressing Smad3 (lanes
3 and 4) or empty vector (lanes 1 and
2) were separated by electrophoresis and probed with
antibodies to Smad3 (upper panel) or actin (lower
panel). C, fibroblasts were treated with TGF-
and/or
IFN-
for 2 h. At the end of the incubation period, cells were
fixed and processed for immunocytochemistry, and Smad3 (left
panel) or Smad4 (right panel)-specific
immunofluorescence was detected by confocal microscopy, as described
under "Materials and Methods." The percentage of fibroblasts
showing predominantly nuclear Smad localization, shown in the
upper right panels, was determined by counting 100 cells.
Representative photomicrographs are shown. Results were essentially
identical when incubation with IFN-
/TGF-
was continued for 4 h.
treatment of fibroblasts caused rapid Stat1 nuclear
accumulation (Fig. 2); and increase in its DNA binding activity (62).
To directly determine the role of Stat1
in mediating repression of
TGF-
-induced COL1A2 transcription by IFN-
, the response of Jak1-deficient U4A cells was examined. These cells are a
genetically defined system commonly used to delineate IFN-
signaling
(55). Treatment of U4A cells with IFN-
had little effect on
COL1A2 promoter activity in the presence of TGF-
(Fig. 5). In contrast, U4A cells rescued with
stable expression of exogenous Jak1 (U4A/Jak1)
demonstrated normal Stat1
-mediated responses, and IFN-
caused
>70% decrease in TGF-
-stimulated CAT activity. As expected, the
activity of the GAS-driven reporter was markedly up-regulated by
IFN-
in the parental, but not in the signaling defective, cells
(data not shown). We therefore conclude that Stat1
activity is
required for IFN-
abrogation of TGF-
-induced, Smad3-mediated
COL1A2 transactivation. Because Stat1 activation is rapid
and precedes repression of COL1A2, it is likely to represent an early step in a series of events culminating in transcriptional repression.
View larger version (21K):
[in a new window]
Fig. 5.
IFN- repression of
TGF-
-induced transcription requires endogenous
activation of Stat1
.
Jak1-deficient U4A cells and U4A/Jak1 cells were
transfected with 772COL1A2/CAT. Following 48 h
incubation in the presence of TGF-
with or without IFN-
, cells
were harvested and CAT activities were determined. The results shown
represent the mean of two determinants; *, p < 0.05.
and IFN-
Signals Are Integrated at the Level of p300/CBP
Coactivators--
Certain responses mediated by IFN-
/Stat1
and
TGF-
/Smad3 pathways are critically dependent on p300/CBP
coactivators (9, 36-42, 45). We reasoned, therefore, that the
antagonistic effect of IFN-
on Smad-mediated TGF-
transactivation
of COL1A2 could result from competition between Smad3 and
IFN-
-activated signal transducers such as Stat1
for interaction
with a limiting cellular pool of p300/CBP. Consistent with this
possibility, we found that overexpression of p300 in fibroblasts
rescued TGF-
stimulation of COL1A2 promoter activity
(Fig. 6A) and endogenous
collagen accumulation (Fig. 6B) in the presence of
antagonistic IFN-
. In contrast, a mutant form of p300 defective in
histone acetyl- transferase activity failed to relieve IFN-
repression (data not shown); indicating that modulation of
COL1A2 transcription involved the histone acetylase function
of p300. p300 enhanced transactivation of a minimal GAS
promoter by low concentrations (10 units/ml) of IFN-
, suggesting a
significant in vivo functional interaction between p300 and
activated endogenous Stat1
in fibroblasts (Fig. 6C). To
examine if the antagonistic effect of IFN-
on TGF-
-stimulated COL1A2 transactivation could be due to down-regulation of
cellular p300 levels, immunoblotting was performed. The results showed that the pool of endogenous p300 was slightly increased in
IFN-
-treated fibroblasts, indicating that IFN-
interfered with
the function, and not the amount, of p300 in these cells (Fig.
6D). Consistent with this notion, we also found that IFN-
had no detectable effect on the levels or subcellular distribution of
endogenous p300 (data not shown). Taken together, these results
suggested that p300 was present in limiting amounts in fibroblasts, and
the ability of IFN-
to inhibit Smad3 transactivation resulted from
competition for p300 by Stat1
or other transcription factors induced
by IFN-
.
View larger version (33K):
[in a new window]
Fig. 6.
Overexpression of p300 relieves the
antagonistic effect of IFN- on
COL1A2 stimulation. A, fibroblasts
were co-transfected with the plasmid expressing p300, along
with 772COL1A2/CAT. Following 48 h incubation with or
without TGF-
and/or IFN-
, CAT activities were determined. Results
shown represent the mean of several independent experiments.
B, levels of Type I collagen in fibroblasts transfected with
p300 expression vector. Whole cell lysates were prepared
following a 48-h incubation with IFN-
and/or TGF-
, and following
gel electrophoresis, proteins were subjected to Western blotting using
antibodies to Type I collagen and actin. C, fibroblasts were
co-transfected with the plasmid expressing p300, along with
a minimal construct containing the consensus GAS linked to
the CAT reporter gene. Following 6 h incubation with
(closed bars) or without (open bars) IFN-
(10 units/ml), CAT activities were determined. D, modulation of
cellular p300 levels by IFN-
. Whole cell lysates were prepared
following a 48-h incubation of confluent fibroblasts with the indicated
concentrations of IFN-
, and examined by immunoblot using antibodies
to p300 and actin.
alone, or together with
IFN-
for 30 min, a time point sufficient for induction of p300
interaction with both Stat1 and Smad3 (36, 45). At the end of the
incubations, whole cell lysates from fibroblasts transfected with
p300 expression plasmid and treated with IFN-
and/or
TGF-
for 30 min were immunoprecipitated with p300 antibody and
following electrophoresis, examined by immunoblot with antibodies to
Smad3. As shown in Fig. 7A,
the TGF-
-induced interaction of p300 with endogenous Smad3 appeared to be modestly reduced upon treatment of the fibroblasts with IFN-
.
Next, the interaction of Stat1
with p300 was examined. Immunoprecipitated proteins were examined by immunoblot with antibodies to Stat1
or tyrosine-phosphorylated Stat1
. As shown in Fig. 7B, IFN-
strongly enhanced the interaction of p300 with
tyrosine-phosphorylated Stat1
, and this interaction was not altered
by TGF-
. These results demonstrate that in primary fibroblasts, p300
interacted with both activated Smad3 and activated Stat1
in
vivo; in a ligand-dependent manner. Whereas IFN-
reduced the interaction of p300 with endogenous Smad3, TGF-
was
unable to modulate the association of p300 with activated Stat1
.
View larger version (38K):
[in a new window]
Fig. 7.
Ligand-induced interaction of p300 with
activated cellular Smad3 or Stat1 in
vivo. Confluent fibroblasts were transfected with
p300 expression plasmids. Following incubation in the
presence or absence of IFN-
or TGF-
for 30 min, whole cell
lysates were prepared and immunoprecipitated with antibodies to p300.
Equal amounts of immunoprecipitated proteins were analyzed by
immunoblot with antibodies to: A, Smad3 and p300; or
B, tyrosine-phosphorylated Stat1
or Stat1
. Last
lane, control IgG.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and inhibited by
IFN-
(47-51). Therefore, the intracellular cross-talk between the
signaling pathways activated by these two functionally antagonistic
cytokines is of substantial interest.
and IFN-
signaling at the level of the shared cofactors
p300/CBP. Stat1
, a critical transducer of IFN-
responses, is one
of a large group of transcription factors that employ p300/CBP to bring
about their effects on transcription. Interactions between Stat1
and
p300/CBP can be ligand-dependent or constitutive,
indicating that both unphosphorylated monomeric, and phosphorylated
dimeric Stat1
can interact with p300/CBP. We have previously
demonstrated that in primary fibroblasts, endogenous p300/CBP is
required by activated Smad3 to stimulate COL1A2
transcription (42). TGF-
induces direct binding of Smad3 to p300/CBP
(36-42). In fibroblasts exposed to TGF-
and IFN-
simultaneously,
activated Stat1
and Smad3 are therefore likely to compete with each
other for interaction with p300/CBP. Because p300/CBP is present in
limiting amounts in these cells, the net effect of simultaneous
signaling by the two antagonistic cytokines on target gene
transcription appears to be determined by the levels of the
coactivators, and the relative affinity of their interaction with Smad3
and IFN-
signal transducers. Although we cannot exclude the
possibility that integration of antagonistic signals at the level of
p300 involves direct inhibition of p300 function by IFN-
/Stat1
,
rather than its simple sequestration, the functional synergism between
p300/CBP and IFN-
shown in Fig. 6C suggests that this is
not the case. Further studies to examine the effect of IFN-
on
p300/CBP histone acetyltransferase activity are currently in
progress. The model for integration of antagonistic transcriptional
signals through their competition for nuclear coactivators is
illustrated in Fig. 8. This model
proposed here is reminiscent of the regulation of the scavenger
receptor gene by IFN-
and macrophage colony-stimulating factor via
p300/CBP (45). The well documented antagonistic effect of nuclear
receptors on AP-1-mediated signaling likewise is ascribed to
competition for limiting amounts of cellular cofactors (67).
Furthermore, while this paper was under review, several reports have
demonstrated that antagonistic regulation of Smad-mediated
transcriptional responses by TNF-
is also mediated through
competition for cellular p300 (68, 69).
View larger version (22K):
[in a new window]
Fig. 8.
Antagonistic regulation of COL1A2
transcription in primary fibroblasts by TGF-
(+) and IFN-
(
). The two
cytokines exert opposing effects on gene expression through competition
between Smads and STAT1 for limiting amounts of the shared cellular
coactivators p300/CBP.
In contrast, a distinct model has been proposed to account for the
suppression of other TGF--induced transcriptional responses by
IFN-
(65) or TNF-
(66). In examining the antagonistic regulation
of 3TP-lux activity by TGF-
and IFN-
, Ulloa et
al. (65) concluded that IFN-
suppression of Smad-mediated
responses was mediated by endogenous Smad7 functioning as autocrine
inhibitor of TGF-
signaling. Similarly, the opposing activities of
TNF-
on TGF-
-induced transactivation of 3TP-lux or
SBE4-luc were shown to be mediated by Smad7 (66). In the
present studies using primary skin fibroblasts, we were unable using a
variety of approaches to detect induction of Smad7 expression by
IFN-
; furthermore, down-regulation of endogenous Smad7 levels by
antisense failed to abrogate the inhibitory effects of IFN-
on
TGF-
-stimulated transcription in these cells. The findings suggest
that expression and autoregulatory function of ligand-induced Smad7 may
show cell-lineage specific differences.
Two additional lines of evidence are provided here to exclude the role
of antagonistic Smad7 in mediating suppression of TGF- responses by
IFN-
. First, we demonstrated that IFN-
was capable of abrogating
COL1A2 transcriptional stimulation induced by Smad3, indicating that the antagonistic cross-talk between TGF-
and IFN-
occurred downstream of the ligand-bound TGF-
receptors. Furthermore,
treatment of the fibroblasts with IFN-
did not prevent TGF-
-induced nuclear accumulation of pathway-restricted Smads, indicating that, in distinct contrast to the endogenous Smad7 autoinhibition model proposed by Ulloa et al. (65), negative regulation of Smad signaling by IFN-
in fibroblasts occurred distal
to Smad3 activation or Smad3-Smad4 complex nuclear translocation. Our
observations are consistent with a model whereby competition of
regulated transcription factors for p300/CBP interaction, which occurs
within the nucleus, but not with Smad7-mediated blockade of the
activation of pathway-restricted Smads by TGF-
receptors. Finally,
endogenous Smad7 induction by TGF-
abrogated the stimulation of
COL1A2 promoter activity (15), indicating that antagonistic Smad7 serves to limit positive regulation of transcription, rather than
mediating ligand-induced negative regulation, in primary fibroblasts.
In summary, the present results provide compelling evidence for the
role of p300/CBP in antagonistic regulation of COL1A2 transcription by TGF- and IFN-
. The results indicate that in normal skin fibroblasts, the intracellular signaling pathways triggered
by TGF-
and IFN-
are integrated at the nuclear level, and appear
to involve a competition between activated Stat1
and Smad3 for
interactions with limiting amounts of cellular p300/CBP. Further
characterization of the molecular basis by which IFN-
inhibits
SMAD-mediated transcriptional activity in fibroblasts will facilitate
the design of specific inhibitors of Smad signaling, which may be
therapeutically useful in scleroderma and other fibrotic disease
characterized by excessive TGF-
responses. Taken together with
recent reports, our observations suggest that antagonistic signals
regulating cell functions are integrated by distinct mechanisms that
are context- and lineage-specific.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank O. Colamonici (University of
Illinois) for the U4A/neo and U4A/Jak1 cells; P. ten Dijke (Ludwig
Institute for Cancer Research, Uppsala, Sweden) for the antisense
Smad7 plasmid; J-M. Gauthier (Glaxo Welcome, France) for the
CAGA-COL1A2/CAT plasmid; G. Sen (Cleveland Clinic Foundation
Research Institute) for the GAS-tk-CAT plasmid; L. Zawel
(Johns Hopkins University) for the pSBE4-luc plasmid; H. Lodish (Whitehead Institute) for the Smad3 expression
plasmid; J. Boyes (Duke University) for the p300 plasmid; and J. Ting (University of North Carolina) for the
-DR152-CAT plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR42309 (to J. V.) and AR46390 (to A. K. G.).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: Section of
Rheumatology (M/C733), University of Illinois Chicago College of
Medicine, 1158 Molecular Biology Research Bldg., 900 S. Ashland Ave.,
Chicago, IL 60607. Tel.: 312-413-9310; Fax: 312-413-9271; E-mail:
jvarga@uic.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M004709200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
CBP, CREB-binding protein;
IFN, interferon;
GAS,
-activated sites;
bp, base pair(s);
CAT, chloramphenicol acetyltransferase;
SBE, Smad-binding element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kingsley, D. M. (1994) Genes Dev. 8, 133-146[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Ignotz, R. A.,
Endo, T.,
and Massague, J.
(1987)
J. Biol. Chem.
262,
6443-6446 |
3. |
Jimenez, S. A.,
Varga, J.,
Olsen, A.,
Li, L.,
Diaz, A.,
Herhal, J.,
and Koch, J.
(1994)
J. Biol. Chem.
269,
12684-12691 |
4. |
Inagaki, Y.,
Truter, S.,
and Ramirez, F.
(1994)
J. Biol. Chem.
269,
14828-14834 |
5. |
Chung, K-Y.,
Agarwal, A.,
Uitto, J.,
and Mauviel, A.
(1996)
J. Biol. Chem.
271,
3272-3278 |
6. |
Border, W. A.,
and Noble, N. A.
(1994)
N. Engl. J. Med.
331,
1286-1292 |
7. | Derynck, R., Zhang, Y., and Feng, X-H. (1998) Cell 95, 737-740[Medline] [Order article via Infotrieve] |
8. | Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J. (1996) Nature 383, 832-836[CrossRef][Medline] [Order article via Infotrieve] |
9. | Zhang, Y., Feng, X-H., Wu, R-Y., and Derynck, R. (1996) Nature 383, 168-171[CrossRef][Medline] [Order article via Infotrieve] |
10. | Wu, R. Y., Zhang, Y., Feng, X-H., and Derynck, R. (1997) Mol. Cell. Biol. 17, 2521-2528[Abstract] |
11. | Macías-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224[Medline] [Order article via Infotrieve] |
12. | 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] |
13. | Nakao, A., Afrakhte, M., Morén, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, N. E., Heldin, C. H., and ten Dijke, P. (1997) Nature 389, 631-635[CrossRef][Medline] [Order article via Infotrieve] |
14. | Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., Richardson, M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D. (1997) Cell 89, 1165-1173[Medline] [Order article via Infotrieve] |
15. |
Chen, S-J.,
Yuan, W.,
Mori, Y.,
Levenson, A.,
Trojanowska, M.,
and Varga, J.
(1999)
J. Invest. Dermatol.
112,
49-57 |
16. | 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] |
17. |
Dennler, S.,
Itoh, S.,
Vivien, D.,
ten Dijke, P.,
Huet, S.,
and Gauthier, J. M.
(1998)
EMBO J.
17,
3091-3100 |
18. |
Jonk, L. J. C.,
Itoh, S.,
Heldin, C.-H.,
ten-Dijke, P.,
and Kruijer, W.
(1998)
J. Biol. Chem.
273,
21145-21152 |
19. | 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] |
20. |
Hua, X.,
Miller, Z. A.,
Wu, G.,
Shi, Y.,
and Lodish, H. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13130-13135 |
21. | Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G., and Whitman, M. (1997) Nature 389, 85-89[CrossRef][Medline] [Order article via Infotrieve] |
22. | Zhang, Y., Feng, X-H., and Derynck, R. (1998) Nature 394, 909-913[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Liberati, N. T.,
Datto, M. B.,
Frederick, J. P.,
Shen, X.,
Wong, C.,
Rougier-Chapman, E. M.,
and Wang, X. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4844-4849 |
24. |
Hua, X.,
Liu, X.,
Ansari, D. O.,
and Lodish, H. F.
(1998)
Genes Dev.
12,
3084-3095 |
25. |
Moustakas, A.,
and Kardassis, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6733-6738 |
26. |
Yanagisawa, J.,
Yanagi, Y.,
Masuhiro, Y.,
Suzawa, M.,
Watanabe, M.,
Kashiwagi, K.,
Toriyabe, T.,
Kawabata, M.,
Miyazono, K.,
and Kato, S.
(1999)
Science
283,
1317-1321 |
27. | Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve] |
28. | Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve] |
29. | Turner, B. M. (1998) Cell. Mol. Life Sci. 54, 21-31[CrossRef][Medline] [Order article via Infotrieve] |
30. | Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Perkins, N. D.,
Felzien, L. K.,
Betts, J. C.,
Leung, K.,
Beach, D. H.,
and Nabel, G. J.
(1997)
Science
275,
523-527 |
32. | Bannister, A. J., Oehler, T., Wilhelm, D., Angel, P., and Kouzarides, T. (1995) Oncogene 11, 2509-2514[Medline] [Order article via Infotrieve] |
33. | Bannister, A. J., and Kouzarides, T. (1995) EMBO J. 14, 4758-4762[Abstract] |
34. | Bhattacharya, S., Eckner, R., Grossman, S., Oldread, E., Arany, Z., D'Andrea, A., and Livingston, D. M. (1996) Nature 383, 344-347[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Nakashima, K.,
Yanagisawa, M.,
Arakawa, H.,
Kimura, N.,
Hisatsune, T.,
Kawabata, M.,
Miyazono, K.,
and Taga, T.
(1999)
Science
284,
479-482 |
36. |
Janknecht, R.,
Wells, N. J.,
and Hunter, T.
(1998)
Genes Dev.
12,
2114-2119 |
37. |
Feng, X-H.,
Zhang, Y.,
Wu, R. Y.,
and Derynck, R.
(1998)
Genes Dev.
12,
2153-2163 |
38. |
Shen, X.,
Hu, P. P.,
Liberati, N. T.,
Datto, M. B.,
Frederick, J. P.,
and Wang, X. F.
(1998)
Mol. Biol. Cell
9,
3309-3319 |
39. | Nishihara, A., Hanai, J. I., Okamoto, N., Yanagisawa, J., Kato, S., Miyazono, K., and Kawabata, M. (1998) Gene Cells 3, 613-623 |
40. |
Pouponnot, C.,
Jayaraman, L.,
and Massague, J.
(1998)
J. Biol. Chem.
273,
22865-22868 |
41. |
Topper, J. N.,
Dichiara, M. R.,
Brown, J. D.,
Williams, A. J.,
Falb, D.,
Collins, T.,
and Gimbrone, M. A., Jr.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9506-9511 |
42. | Ghosh, A. K., Yuan, W., Mori, Y., and Varga, J. (2000) Oncogene 19, 3546-3555[CrossRef][Medline] [Order article via Infotrieve] |
43. | Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997) Annu. Rev. Immunol. 15, 749-795[CrossRef][Medline] [Order article via Infotrieve]. |
44. | Darnell, J. E., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1419[Medline] [Order article via Infotrieve] |
45. |
Horvai, A. E.,
Xu, L.,
Korzus, E.,
Brard, E.,
Kalafus, D.,
Mullen, T. M.,
Rose, D. W.,
Rosenfeld, D. W.,
and Glass, C. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1074-1079 |
46. |
Zhang, J. J.,
Vinkemeier, U.,
Gu, W.,
Chakravarti, D.,
Horvath, C. M.,
and Darnell, J. E., Jr.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
15092-15096 |
47. | Kähäri, V.-M., Chen, Y. Q., Su, M. W., Ramirez, F., and Uitto, J. (1990) J. Clin. Invest. 86, 1489-1495[Medline] [Order article via Infotrieve] |
48. | Varga, J., Olsen, A., Herhal, J., Constantine, G., Rosenbloom, J., and Jimenez, S. A. (1990) Eur. J. Clin. Invest. 20, 487-493[Medline] [Order article via Infotrieve] |
49. | Yufit, T., Vining, V., Wang, L., Brown, R. R., and Varga, J. (1995) J. Invest. Dermatol. 105, 388-393[Abstract] |
50. | Higashi, K., Kouba, D. J., Song, Y. J., Uitto, J., and Mauviel, A. (1998) Matrix Biol. 16, 447-456[CrossRef][Medline] [Order article via Infotrieve] |
51. | Yuan, W., Yufit, T., Li, L., Mori, Y., Chen, S. J., and Varga, J. (1999) J. Cell. Physiol. 179, 97-108[CrossRef][Medline] [Order article via Infotrieve] |
52. | Ala-Aho, R., Johansson, N., Grenman, R., Fusenig, N. E., Lopez-Otin, C., and Kähäri, V. M. (2000) Oncogene 19, 248-257[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Sharma, B.,
and Iozzo, R. V.
(1998)
J. Biol. Chem.
273,
4642-4646 |
54. |
Bauvois, B.,
Rouillard, D.,
Sanceau, J.,
and Wietzerbin, J.
(1992)
J. Immunol.
148,
3912-3919 |
55. | Muller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, O., Harpur, A. G., Barbieri, G., Witthuhn, B. A., and Schindler, C. (1993) Nature 366, 129-135[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Ihn, H.,
LeRoy, E. C.,
and Trojanowska, M.
(1997)
J. Biol. Chem.
272,
24666-24672 |
57. | Boyes, J., Byfield, P., Nakatani, Y., and Ogryzko, V. (1998) Nature 396, 594-598[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Liu, X.,
Sun, Y.,
Constantinescu, S. N.,
Karam, E.,
Weinberg, R. A.,
and Lodish, H. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10669-10674 |
59. | Afrakhte, M., Moren, A., Jossan, S., Itoh, S., Sampath, K., Westermark, B., Heldin, C. H., Heldin, N. E., and ten Dijke, P. (1998) Biochem. Biophys. Res. Commun. 249, 505-511[CrossRef][Medline] [Order article via Infotrieve] |
60. | Moses, H., Panek, R. B., Benveniste, E. N., and Ting, JP-Y. (1992) J. Immunol. 148, 3642-3651 |
61. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
62. | Yuan, W., Collado-Hidalgo, A., Yufit, T., Taylor, M., and Varga, J. (1998) J. Cell. Physiol. 177, 174-186[CrossRef][Medline] [Order article via Infotrieve] |
63. |
Venkataraman, C.,
Leung, S.,
Salvekar, A.,
Mano, H.,
and Schindler, U.
(1999)
J. Immunol.
162,
4053-4061 |
64. |
Dickensheets, H.,
Venkataraman, C.,
Schindler, U.,
and Donnelly, R. P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10800-10805 |
65. | Ulloa, L., Doody, J., and Massague, J. (1999) Nature 397, 710-713[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Bitzer, M.,
von Gersdorff, G.,
Liang, D.,
Dominguez-Rosales, A.,
Beg, A.,
Rojkind, M.,
and Bottinger, E.
(2000)
Genes Dev.
14,
187-197 |
67. | Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[Medline] [Order article via Infotrieve] |
68. |
Verrecchia, F.,
Pessah, M.,
Atfi, A.,
and Mauviel, A.
(2000)
J. Biol. Chem.
275,
30226-30231 |
69. | Nagarajan, R. P., Chen, F., Li, W., Vig, E., Harrington, M. A., Nakshatri, H., and Chen, Y. (2000) Biochem. J. 348, 591-596[CrossRef][Medline] [Order article via Infotrieve] |