From INSERM U532, Hôpital Saint-Louis, 75475 Paris, France and the § Department of Dermatology and
Cutaneous Biology, Thomas Jefferson University, Philadelphia
Pennsylvania
Received for publication, January 26, 2001
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ABSTRACT |
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Despite major advances in the
understanding of the intimate mechanisms of transforming growth
factor- Members of the TGF- TGF- Although tremendous progress has been made over the past few years in
the understanding of the molecular processes underlying TGF- Despite the fundamental role played by TGF- Cell Cultures and Reagents--
Human dermal fibroblasts and
Smad3 Differential Hybridization of AtlasTM Human cDNA
Expression Arrays--
Total RNA from control and TGF- Plasmid Constructs--
Several ECM gene promoter/reporter gene
constructs were used to examine the role played by TGF- Transient Cell Transfection and CAT Reporter
Assays--
Transient cell transfections were performed with the
calcium phosphate/DNA co-precipitation procedure. CAT activity was
measured using [14C]chloramphenicol as substrate (40)
followed by thin layer chromatography and quantitation with a phosphorimager.
Effects of TGF-
Because our main aim was to identify immediate-early targets of the
Smad pathway, genes induced as early as 30 min post-TGF-
In the presence of curcumin, an inhibitor of JNK activity (42), only
three genes among the 58 identified above and belonging to clusters
1-3 were not stimulated by TGF- Effects of TGF- Dominant-Negative Smad3 and Inhibitory Smad7 Expression Block
TGF- COL1A1 and COL5A2, but Not COL6A2, Are Direct Smad
Targets--
Because both COL6A1 and COL6A3 were characterized as
direct Smad targets (see above), we wanted to determine whether COL6A2, which encodes the
mRNA steady-state levels of COL1A1, which encodes the Absence of COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, COL6A3, and
TIMP-1 Promoter Transactivation by TGF-
The implication of TGF-
In conclusion, using cell matrix interaction-specific commercial
cDNA microarrays we have identified 58 immediate-early targets for
TGF- (TGF-
) signaling through the Smad pathway, little
progress has been made in the identification of direct target genes. In
this report, using cDNA microarrays, we have focussed our attention
on the characterization of extracellular matrix-related genes rapidly
induced by TGF-
in human dermal fibroblasts and attempted to
identify the ones whose up-regulation by TGF-
is Smad-mediated. For
a gene to qualify as a direct Smad target, we postulated that it had to
meet the following criteria: (1) rapid (30 min) and significant
(at least 2-fold) elevation of steady-state mRNA levels upon
TGF-
stimulation, (2) activation of the promoter by both exogenous
TGF-
and co-transfected Smad3 expression vector, (3) up-regulation
of promoter activity by TGF-
blocked by both dominant-negative Smad3
and inhibitory Smad7 expression vectors, and (4) promoter
transactivation by TGF-
not possible in
Smad3
/
mouse embryo fibroblasts.
Using this stringent approach, we have identified COL1A2, COL3A1,
COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as
definite TGF-
/Smad3 targets. Extrapolation of this approach to other
extracellular matrix-related gene promoters also identified COL1A1 and
COL5A2, but not COL6A2, as novel Smad targets. Together, these results
represent a significant step toward the identification of novel,
early-induced Smad-dependent TGF-
target genes in fibroblasts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1
superfamily (activin, bone morphogenic proteins, TGF-
s, and
decapentaplegic) are multifunctional cytokines that control
various aspects of cell growth and differentiation and play an
essential role in embryonic development, tissue repair, or immune
homeostasis (1, 2). In addition, TGF-
is the prototypic fibrogenic
cytokine, enhancing extracellular matrix (ECM) gene expression and
down-regulating that of matrix-degrading enzymes. Increased expression
of TGF-
is often associated with fibrotic states and abnormal
accumulation of ECM proteins in affected tissues (3-6). The TGF-
s
signal via serine/threonine kinase transmembrane receptors, which
phosphorylate cytoplasmic mediators of the Smad family (7-9). The
ligand-specific Smad1, Smad2, Smad3, and Smad5 interact directly with,
and are phosphorylated by, activated TGF-
receptors type I. Smad1
and Smad5 are specific for bone morphogenic proteins, whereas
Smad2 and Smad3 can be activated by both TGF-
and activin receptors.
Receptor-activated Smads are kept in the cytoplasm in the basal state
bound to the protein SARA (Smad anchor for
receptor activation) (10). Upon phosphorylation at their SSXS carboxyl-terminal motif, they are released
from SARA and form heteromeric complexes with Smad4, a common mediator for all Smad pathways. The resulting Smad heterocomplexes are then
translocated into the nucleus where they activate target genes, binding
DNA either directly or in association with other transcription factors.
Members of the third group of Smads, the inhibitory Smads, Smad6 and
Smad7, prevent phosphorylation and/or nuclear translocation of
receptor-associated Smads (7-9).
also initiates other signaling pathways, such as the
stress-activated protein kinase/c-Jun amino-terminal kinase
(JNK) pathway (11). This intracellular signaling proceeds
through sequential activation of a mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase (MEKK1), a
mitogen-activated protein kinase kinase (MKK4 or MKK7), and a
mitogen-activated protein kinase, JNK. JNK then translocates into the
nucleus where it phosphorylates several transcription factors including
c-Jun, ATF-2, and Elk-1 (12), leading to specific transcriptional responses.
signal
propagation by the Smad cascade, thus far, relatively few genuine
mammalian Smad gene targets have been identified. Specifically, at
present, only about a dozen genes are known to contain Smad-responsive
regions, binding Smad complexes directly or indirectly. These genes
include, in order of characterization as Smad targets, type VII
collagen (COL7A1) (13), plasminogen activator inhibitor-1 (14), the
cyclin-dependent kinase inhibitor p21 (15), JunB (16), and
more recently, COL1A2 (17), c-Jun (18), immunoglobulin germline C
(19), Smad7 (20), human germline IgA (21), platelet-derived growth
factor-
(22),
5 integrin (23), apoCIII (24), and the
cyclin-dependent kinase inhibitor p15 (25). Interestingly,
to date, all Smad gene targets identified downstream of TGF-
are
Smad3-, not Smad2-, dependent.
in ECM remodeling and as
a fibrogenic factor, little is known about the role of Smad signaling
in ECM gene expression. In this report, we have used complementary
techniques, differential hybridization of cDNA microarrays together
with precise gene promoter analyses, to search for novel fibroblast
Smad targets. This approach allowed us to characterize six novel Smad
targets, COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1, and to
propose an additional list of 49 immediate-early TGF-
target genes
whose activation by TGF-
is rapid and does not require either
protein neo-synthesis or JNK activity, therefore representing potential novel Smad targets.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/
mouse embryo fibroblasts (26, 27), a
kind gift from Drs. A. B. Roberts and C. Deng, National Cancer
Institute, NIH, Bethesda, MD, were grown in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated fetal calf
serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin, 50 µg/ml streptomycin-G, and 0.25 µg/ml FungizoneTM). Human recombinant TGF-
1 was from R & D Systems, Inc. (Minneapolis, MN). Curcumin and cycloheximide were purchased from Sigma.
-treated
fibroblasts was obtained using an RNeasy kit (Qiagen) and treated with
DNase I to avoid genomic DNA contamination of reverse transcription
reactions. Radioactive cDNA synthesis was carried out as described
in the AtlasTM cDNA expression arrays user manual
(CLONTECH, San Diego, CA). Equal amounts of
33P-radiolabeled cDNAs (107 cpm) from
control and TGF-
-treated fibroblast RNA samples were hybridized in
parallel to AtlasTM human cell interaction cDNA expression arrays
(catalog number 7746-1; CLONTECH) for
18 h at 68 °C. The filters were then washed four times in 2×
SSC and 1% SDS for 30 min at 68 °C and twice in 0.1× SSC and 0.5%
SDS at 68 °C, according to the manufacturer's protocol. Membranes
were then exposed to Eastman Kodak Co. phosphor screens for 3 days.
Hybridization signals were quantified with a Storm 840 phosphorimager
using ImageQuant software (Amersham Pharmacia Biotech) and normalized
against glyceraldehyde-3-phosphate dehydrogenase mRNA levels in the
same samples. Significant modulation of gene expression was set
arbitrarily to 2-fold.
and Smad3 in
their transcriptional activity. The human COL1A2, COL6A1, COL6A2,
COL7A1, and decorin promoter constructs have been described previously
(28-32). Human COL6A3 promoter was cloned recently in our
laboratory.2 Human COL1A1
(33), mouse COL3A1 (34), human COL5A2 (35), and human TIMP-1 (36)
promoter constructs were kind gifts from Drs. Sergio A. Jimenez
(Thomas Jefferson University, Philadelphia, PA),
Benoit de Crombrugghe (M. D. Anderson Cancer Center, Houston, TX),
Fransesco Ramirez (Mount Sinai Medical Center, New York, NY), and
Dylan R. Edwards (University of Calgary, Calgary, Alberta, Canada).
Full-length Smad3 and carboxyl-terminally truncated dominant-negative Smad3 (Smad3
C) expression vectors (kind gifts from
Drs. Anita B. Roberts and Rober J. Lechleider, National Cancer
Institute, NIH, Bethesda, MD and Rik Derynck, University of California
at San Francisco, San Francisco, CA, respectively) have been described previously (37, 38). Smad7 expression vector (39) was a kind gift from
Dr. Peter ten Dijke, Netherlands Kanker Instituut, Amsterdam, Netherlands.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
on Fibroblast ECM-related Gene Expression
Profiles as Measured by cDNA Microarray Analysis--
The
technique of differential hybridization of cDNA expression arrays
was used to identify differences in the expression pattern of 265 known
ECM-related genes between control and TGF-
-treated fibroblasts.
Because Smad activation and nuclear translocation occurs within minutes
and Smad-DNA complexes can be observed as early as 10 min after TGF-
addition into fibroblast culture medium (13), we focused our attention
on early time points, to determine which genes are activated rapidly by
TGF-
, as opposed to secondary gene activation that may involve
protein/transcription factor neo-synthesis. At each of the
time points tested (30, 60, 120, and 240 min), RNA was extracted
from both control and TGF-
-treated fibroblast cultures, and
differential hybridization of cDNA arrays was performed. Among the
265 genes whose probe sets are represented onto the AtlasTM cell
interactions cDNA arrays used in these experiments, 77 of them
showed no significant hybridization signal in either control or
TGF-
-treated cultures at any of the time points tested (not shown).
Among the 188 genes detected, 90 showed no or little alteration in
their expression levels upon TGF-
treatment. The remaining 98 genes,
modulated by TGF-
, were classified into clusters, based upon the
temporal profile of their activation (Fig.
1). Clusters 1-3 contain 58 genes whose
expression is strongly up-regulated 30 min after TGF-
addition and
keeps increasing with time (cluster 1), reaches a plateau (cluster 2),
or returns rapidly to basal level (cluster 3). Clusters 4-6 comprise
genes whose expression is delayed; their expression is not noticeably
up-regulated by TGF-
at the 30-min time point and then follows
various patterns of temporal regulation. The complete list of
genes contained within these clusters is provided in Table
I.
View larger version (20K):
[in a new window]
Fig. 1.
Average expression profiles for each gene
cluster. Six clusters were identified showing different induction
kinetics by TGF- . Values are the mean -fold induction by TGF-
of
all genes in each cluster.
List of genes classified in clusters according to their induction
kinetics by TGF-
in two independent
experiments. They are classified in six clusters according to the
kinetics of their induction (see Fig. 1). GenBankTM accession numbers,
gene names, and categories for classification are provided, as supplied
by Clontech. Note that clusters 1-3 contain genes whose expression is
significantly upregulated by TGF-
after 30 min.
addition to
the cultures were further studied in experiments in which on-going
protein synthesis is blocked by cycloheximide. Analysis of gene
expression by differential hybridization of cDNA arrays indicated
that, at the 30-min time point, a similar set of 58 genes was induced
by TGF-
in the presence or absence of cycloheximide, consistent with
a transcriptional response not requiring on-going protein synthesis,
such as expected from direct Smad targets. It should be noted that a
broad increase in gene expression induced by cycloheximide alone was
also observed (not shown), a phenomenon that has been previously
described (41).
after 30 min, fibronectin, perlecan, and closely related low density lipoprotein receptor (43).
Interestingly, it has have been shown previously that fibronectin gene
activation by TGF-
is a JNK-dependent mechanism that
does not require the Smad pathway (44), consistent with the inhibitory
effect of curcumin observed in our experiments. Regarding perlecan, we
have shown previously that its up-regulation by TGF-
is mediated by
transcription factor NF-1 and not by Smads (45). Inversely, we found
that genes previously identified as Smad targets, plasminogen activator
inhibitor-1 (14), COL1A2 (17), and
5 integrin (23) (see Table I and
the Introduction), as well as p21 (15), the latter detected in another
set of experiments using different cDNA microarrays (catalog number
7741-1; CLONTECH, not shown), also belong to these
early-induced gene clusters. Together, these data provide a strong
argument for the specificity of our experimental approach and its
appropriateness for the characterization of early-induced TGF-
/Smad targets.
and Smad3 Overexpression on ECM Promoter/CAT
Reporter Gene Constructs--
We next tried to determine whether the
rapid elevation of steady-state mRNA levels observed for several
ECM-related genes upon TGF-
stimulation, as observed using
differential cDNA array hybridization, resulted from
transcriptional activation at the level of their promoter regions. We
focused our attention on the 5' regulatory regions of COL1A2, COL3A1,
COL6A1, COL6A3, and TIMP-1 genes, which all belong to clusters 1 and 2, corresponding to genes whose expression is enhanced at least two times
by TGF-
within 30 min. In a first set of experiments, TGF-
responsiveness was examined. All promoter constructs tested responded
to exogenous addition of TGF-
by a 3-5-fold elevation of their
activity (Fig. 2A). As a first
approach to determine whether these promoters were sensitive to Smad
activation downstream of TGF-
, co-transfection experiments were
performed in which each ECM promoter/CAT reporter construct was
co-transfected with a Smad3 expression vector. As shown in Fig.
2B, Smad3 overexpression led to significant up-regulation of
each of the promoters tested, indicating that the Smad pathway may be
involved in the TGF-
effect.
View larger version (17K):
[in a new window]
Fig. 2.
Exogenous TGF- and
Smad3 overexpression transactivate the COL1A2, COL3A1, COL6A1, COL6A3,
and TIMP-1 promoters. A, human dermal fibroblast
cultures were transfected with COL1A1, COL3A1, COL6A1, COL6A3, or
TIMP-1 promoter/CAT reporter constructs. 3 h later, TGF-
(10 ng/ml) was added to the medium, and the incubation was continued for
24 h, at which time CAT activity was determined. B,
empty pcDNA3 or Smad3 expression vectors were co-transfected with
the same promoter/reporter constructs as in panel A. 24 h later CAT activity was determined. -Fold induction of promoter
activities by TGF-
(panel A) or Smad3 (panel
B) is shown as the mean ± S.D. of at least three independent
experiments performed in duplicate.
-induced ECM Promoter Activation--
If the Smad cascade is
responsible for the up-regulation of a given gene by TGF-
, then the
expression of either a dominant-negative Smad3 or inhibitory Smad7
should block its activation by TGF-
. To test this hypothesis, all
promoter constructs shown above to respond to both TGF-
and Smad3
overexpression (see Fig. 2) were co-transfected with either Smad3
C
or Smad7 expression vector. In both cases up-regulation of promoter
activity by TGF-
was blocked (Fig. 3),
suggesting that the COL1A2, COL3A1, COL6A1, COL6A3, and TIMP-1 genes
are immediate-early targets of the TGF-
/Smad3/4 signaling cascade.
It may be extrapolated, although it remains to be investigated
precisely, that most genes identified in clusters 1-3 whose mRNA
steady-state levels were elevated at least two times by TGF-
after
30 min, but whose 5'-end regulatory regions were not analyzed
functionally for Smad responsiveness, also represent direct Smad
targets.
View larger version (22K):
[in a new window]
Fig. 3.
Overexpression of dominant-negative Smad3 or
Smad7 prevents COL1A2, COL3A1, COL6A1, COL6A3, and TIMP-1 promoter
up-regulation by TGF- . Human dermal
fibroblast cultures were co-transfected with COL1A1, COL3A1, COL6A1,
COL6A3, or TIMP-1 promoter/CAT reporter constructs, together with empty
pcDNA3, Smad3
C, or Smad7 expression vectors, as indicated.
3 h later, TGF-
(10 ng/ml) was added (+) or not (
) to the
medium, and incubations were continued for 24 h before CAT
activity was determined. Relative promoter activities (mean ± S.D.) of at least three independent experiments performed in duplicate
are shown in the form of bar graphs.
(2) chain of heterotrimeric type VI
collagen, was similarly up-regulated by TGF-
. Interestingly, when a
2.5-kilobase pair COL6A2 promoter fragment (24) was tested in an
identical experimental system, we observed that it did not confer
either TGF-
or Smad3 responsiveness (not shown), suggesting a
differential regulation of the three genes encoding type VI collagen by
TGF-
, where both COL6A1 and COL6A3 are coordinately regulated and
are direct Smad targets, whereas COL6A2 is not. These data differ slightly from previous observations indicating specific up-regulation of COL6A3 but not COL6A1 or COL6A2 by TGF-
, when mRNA
steady-state levels were detected after 48 h of stimulation
(46).
(1)
chain of type I collagen, have been shown previously to be elevated by
TGF-
(47). The corresponding promoter was found to be up-regulated by both exogenous addition of TGF-
and co-transfection of a Smad3 expression vector (not shown). In addition, its activation by TGF-
was blocked by both dominant-negative Smad3 and Smad7 overexpression (not shown), indicating that the COL1A1 promoter is also a Smad target.
Interestingly, these data corroborate the previously described coordinate regulation of COL1A1 and COL1A2 (48-50) and indicate transcriptional coordination by TGF-
, orchestrated by
Smad-dependent mechanisms. Identical results were obtained
with the
(2) type V collagen gene (COL5A2) promoter, indicating that
the latter is also a direct Smad target (not shown).
in Smad3
/
Mouse Embryo Fibroblasts--
To ascertain the role played by Smad3 in
the transactivation of the ECM-related promoters identified above,
transient cell transfection experiments were carried out using either
wild-type or Smad3
/
mouse embryo fibroblasts
(26, 27). As expected from the data presented above in which either
dominant-negative Smad3 or inhibitory Smad7 expression vectors blocked
TGF-
effect, no TGF-
-driven transactivation of the COL1A1,
COL1A2, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1 promoters was
observed in Smad3
/
mouse embryo fibroblasts.
On the other hand, all promoters add their activity significantly
increased by TGF-
in the corresponding wild-type mouse embryo
fibroblasts (not shown). These results confirm the role played by Smad3
to mediate TGF-
transactivation of these ECM promoters.
in fibrotic processes has long been
suspected (3-6). The demonstration that several genes encoding fibrillar collagens, COL1A1, COL1A2, COL3A1, and COL5A2, are
up-regulated by TGF-
acting directly through the Smad pathway
indicates that the latter is likely to play a key role in the
development of tissue fibrosis. It also suggests that therapeutic
approaches directed toward the Smad cascade may prove useful in the
treatment of fibrotic disorders.
. Only three of these 58 genes had their activation blocked by
curcumin, a selective inhibitor of JNK, a signaling pathway alternative
to the Smad cascade downstream of the TGF-
receptors. These data
suggest that the JNK pathway downstream of the TGF-
receptors likely
affects very few early ECM-related target genes as compared with the
Smad pathway. Using promoter/reporter gene constructs to analyze the
transcriptional responsiveness to the TGF-
/Smad pathway of several
genes identified by differential hybridization of cDNA arrays, we
have formally identified six novel TGF-
/Smad immediate-early gene
targets, namely COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and TIMP-1.
Together with the identification of 49 other immediate-early TGF-
gene targets, this study represents a major leap forward in the
identification of TGF-
/Smad targets.
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ACKNOWLEDGEMENTS |
---|
Charlotte Tacheau provided excellent technical help.
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FOOTNOTES |
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* This work was supported by grants from the Association pour la Recherche contre le Cancer (France; Subvention Libre 9058), INSERM, France (APEX 4X809D), and Electricité de France (Service de Radioprotection) (to A. M.) and by National Institutes of Health Grant AR38912 (to M. L. C.).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: INSERM U532, Institut de Recherche sur la Peau, Pavillon Bazin, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris, Cedex 10, France. Tel.: 33 1 53 72 20 69; Fax: 33 1 53 72 20 51; E-mail: mauviel@chu-stlouis.fr.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100754200
2 R. Z. Zhang and M.-L. Chu, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-(s), transforming growth factor-
(s);
CAT, chloramphenicol
acetyltransferase;
ECM, extracellular matrix;
JNK, Jun amino-terminal
kinase;
TIMP, tissue inhibitor of metalloproteases.
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