From the Department of Pediatrics, Northwestern University Medical School, and Children's Memorial Institute for Education and Research, Chicago, Illinois 60611-3008
Received for publication, July 19, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The mechanism(s) by which Smads mediate and
modulate the transforming growth factor (TGF)- Glomerulosclerosis is a scarring process involving extracellular
matrix (ECM)1 accumulation
and obliteration of glomerular capillaries. It is considered to be the
final pathway leading to the progressive loss of renal function in
several kidney diseases. Mechanical factors such as hyperfiltration and
intraglomerular hypertension, as well as a variety of mediators
including cytokines, growth factors, and eicosanoids derived from
circulating or glomerular cells, have been implicated in initiating or
maintaining sclerosis (1). However, little information is available
regarding the cellular mechanisms by which these factors affect matrix
turnover. Our laboratory has been studying the mechanisms by which
transforming growth factor (TGF)- The Smads are a series of proteins that function downstream from the
serine/threonine kinase receptors of the TGF- Smad3 and Smad4 are able to directly bind to DNA through their
N-terminal MH1 domain (5, 6). Although these studies described two
different consensus sequences for Smad binding (GTCTAGAC, called Smad
binding element or SBE; and AG(C/A)CAGACAC, called CAGA box), both
sequences contain the core motif AGAC, which represents the optimal
binding sequence for Smad3 and Smad4 (6, 7). This motif is present in
the regulatory regions of several TGF- We previously showed that a construct containing the sequence from
In the present study, we sought to further delineate the region
responsible for TGF- Materials--
Reagents were purchased from the following
vendors: active, human recombinant TGF- Cell Culture--
Human mesangial cells were isolated from
glomeruli by differential sieving of minced normal human renal cortex
obtained from anonymous surgery or autopsy specimens. The cells were
grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium,
supplemented with 20% heat-inactivated fetal bovine serum (FBS),
glutamine, penicillin/streptomycin, sodium pyruvate, Hepes buffer, and
8 µg/ml insulin (Life Technologies, Inc.) as described previously (35) and were used between passages 5 and 8.
RNA Isolation and Northern Blot--
Cells were plated in 100-mm
culture dishes (3-5 × 105 cells/dish). The next day,
they were preincubated with mithramycin A (100 mM) for
17 h or curcumin (20 µM) for 30 min before addition of 1 ng/ml TGF- Transient Transfection and Luciferase Assay--
The day before
the transfection, 6.5-8 × 104 cells were seeded in
six-well plates. Eighteen hours later, cells were switched to 1% FBS
medium and transfected with the indicated constructs along with 0.5 µg of CMV-SPORT- Plasmid Constructs--
The 376COL1A2-LUC construct containing
376 bp of the Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays (EMSA)--
Nuclear extracts were prepared from control
and TGF- Preparation of Cell Lysates, Immunoprecipitation, and Western
Blot Analysis--
Cells plated in 150-mm dishes (0.75-1 × 106 cells/plate) were cultured for 3 days. The cells were
switched to medium containing 1% FBS and then treated with 1 ng/ml
TGF- Statistical Analysis--
Statistical differences between
experimental groups were determined by analysis of variance using
InStat 2.03 software program for Macintosh. Values of p < 0.05 were considered significant.
Sp1 Binding Is Necessary for TGF-
To further support these findings and delineate the TGF- Involvement of Sp1 Binding in Smad3-mediated TGF-
We next investigated whether Sp1 binding is required for Smad3-mediated
Similar to the pharmacological inhibition of Sp1 binding, deletion of
the three Sp1 sites (268COL1A2-LUC) decreased the effect of
overexpressed Smad3 (Fig. 5). Deletion of
the CAGA box, leaving the AP-1 motif intact (258COL1A2-LUC), inhibited
TGF- Sp1 and Smad Proteins Bind to the COL1A2 Promoter--
We next
sought to determine whether Sp1 and Smads were able to interact with
the COL1A2 promoter. EMSA were performed with nuclear
extracts from control and TGF- TGF- Functional Interaction between Sp1 and Smad Proteins Is Stimulated
by TGF-
The functional interaction between Smad3 and Sp1 was further examined
by cotransfecting human mesangial cells with the pFR-LUC reporter
construct along with a plasmid encoding for the glutamine-rich transactivation domain B of Sp1 fused to the Gal4 DNA binding domain
(Gal4-Sp1(B), residues 263-542) along with a Smad3 expression vector
or the control empty construct. Additionally, the transcriptional activity of two other fusion proteins were studied: the Gal4-Sp1(B-c), containing residues 421-542 of Sp1, which was shown to activate transcription in Drosophila SL2 and HeLa cells; and the
Gal4-Sp1(B-n), containing residues 263-424, which is inactive (44).
Gal4-Sp1(B) and Gal4-Sp1(B-c) stimulated activity of the pFR-LUC
construct by 5-11-fold (Fig. 9B). TGF- Sp1 and Smad3 Cooperate to Regulate In the present study, we show that Smad proteins and Sp1 interact
together to stimulate human In dermal fibroblasts, the deletion of sequences between Our transient transfection experiments with 5' deletion of the
COL1A2 promoter and analysis of steady-state levels of
mRNA from cells treated with mithramycin A indicate that Sp1
binding is required for TGF- Overexpression of Sp1 alone does not affect basal or TGF- Binding of Sp1 to the COL1A2 promoter sequences between
To our knowledge, this is the first report showing formation of
complexes between endogenous Smad proteins and a member of the Sp
family in nontransformed cells. Sp1 and Smad3/4 associate in the
absence of TGF- Our experiments show that formation of Sp1 and Smad3/4 complexes (Figs.
7 and 8) leads to functional activation of transcription (Figs. 9 and
10). Sp1 cooperates with Smad3 or Smad4 to stimulate transcription in a
Gal4-LUC reporter assay system (Fig. 9A). The ligand-independent stimulation of Gal4-Smad3 by Sp1 is in agreement with the observation of Sp1/Smad3 complexes in the absence of TGF- TGF- Sp1 has been shown to interact directly with components of the TFIID
complex, TAFII110 and TAFII130, via the
C-terminal part of its B domain (44, 65) and those interactions are
thought to participate in the recruitment and/or stabilization of the preinitiation complex at the promoter in eukaryotes. Smad3 and Smad4
have been shown to interact with the coactivators CBP/p300 via their
MH2 domain (24, 26, 28). These coactivators are thought to modulate
transcription by interacting with components of the basal transcription
machinery. They possess histone acetyltransferase activity and could
potentiate transcription by loosening the chromatin structure. Thus,
Sp1 and Smad3/Smad4 could enhance levels of transcription in response
to TGF- In summary, we showed that Sp1 binding is required for Smad3
stimulation of COL1A2 promoter activity and is essential for the human glomerular mesangial cell responsiveness to TGF- signal transduction
pathway in fibrogenesis are not well characterized. We previously
showed that Smad3 promotes
2(I) collagen gene
(COL1A2) activation in human glomerular mesangial
cells, potentially contributing to glomerulosclerosis. Here, we report
that Sp1 binding is necessary for TGF-
1-induced type I collagen
mRNA expression. Deletion of three Sp1 sites (GC box) between
376
and
268 or mutation of a CAGA box at
268/
260 inhibited
TGF-
1-induced
2(I) collagen promoter activity. TGF-
1
inducibility was also blocked by a Smad3 dominant negative mutant.
Chemical inhibition of Sp1 binding with mithramycin A, or deletion of
the GC boxes, inhibited COL1A2 activation by Smad3,
suggesting cooperation between Smad3 and Sp1 in the TGF-
1 response.
Electrophoretic mobility shift assay showed that Sp1 and Smads form
complexes with
283/
250 promoter sequences. Coimmunoprecipitation
experiments demonstrate that endogenous Sp1, Smad3, and Smad4 form
complexes in mesangial cells. In a Gal4-LUC reporter assay system, Sp1
stimulated the TGF-
1-induced transcriptional activity of Gal4-Smad3,
Gal4-Smad4 (266), or both. Using the transactivation domain B of
Sp1 fused to the Gal4 DNA binding domain, we show that, in our system,
the transcriptional activity of this Sp1 domain is not regulated by
TGF-
1, but it becomes responsive to this factor when Smad3 is
coexpressed. Finally, combined Sp1 and Smad3 overexpression induces
marked ligand-independent and ligand-dependent promoter
activity of COL1A2. Thus, Sp1 and Smad proteins form
complexes and their synergy plays an important role in mediating
TGF-
1-induced
2(I) collagen expression in human mesangial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
stimulates ECM accumulation.
Previously, we determined that the Smad pathway plays a role in
activating type I collagen gene expression in human glomerular
mesangial cells.
family to transduce
signal to the nucleus (2-4). Following TGF-
binding to its
receptors, the receptor-regulated Smads (R-Smads), Smad2 and Smad3, are
phosphorylated by the type I receptor and associate with the common
partner, Smad4. The resulting heteromultimer translocates to the
nucleus where it regulates expression of TGF-
target genes (2-4).
The inhibitory Smads, Smad6 and Smad7, may participate in a negative
feedback loop to control TGF-
responses by competitive interaction
with the type I receptor (2-4).
target genes, including those
for PAI-1 (5, 8) JunB (9), c-Jun (10), type VII collagen (11), Smad7
(12, 13), germline Ig
(14, 15), and PDGF-B (16). Several of these
studies showed that mutations of these CAGA boxes in the context of the promoter abrogate TGF-
stimulation. Conversely, multiple copies of
this sequence support ligand inducibility, demonstrating the importance
of Smad3/Smad4 binding sites in mediating TGF-
responsiveness. However, not all target genes of the TGF-
family contain a canonical SBE in their regulatory region (17, 18). Taken together with the fact
that only multiple copies of SBE confer TGF-
inducibility (6) and
that binding of a Smad MH1 to SBE is of low affinity (7), these
observations suggest that the CAGA sequence alone is not sufficient to
support maximal TGF-
-mediated transcriptional activation of target
genes. Indeed, several studies indicate that, in response to
TGF-
/activin, Smads can cooperate with other DNA-binding proteins
such as members of the AP-1 family (10, 19, 20), PEBP2/CBF/AML (14,
15), Fast proteins (18, 21), and TFE3 (8). R-Smads and Smad4 also have
been shown to interact with the coactivators p300/CBP (22-28).
376 to +58 of the human
2(I) collagen gene (COL1A2)
promoter is responsive to TGF-
1 in human mesangial cells and that
overexpression of Smad3 stimulates COL1A2 promoter activity
(29). The TGF-
response element of the
2(I) collagen gene has
been mapped to the sequences located between
340 and
183 from the
transcription start site (30). This region contains three Sp1 binding
sites and one AP-1 consensus sequence. Different groups have implicated either Sp1 or AP-1 as the mediator of TGF-
stimulation of
2(I) collagen gene in dermal fibroblasts (30-32). Between the Sp1 and AP-1
binding sites lies a CAGA box. In gel shift experiments, a
transcriptional complex containing Smad proteins in nuclear extracts
from TGF-
-treated fibroblasts binds to this CAGA box (33). Multiple
copies of this motif conferred TGF-
1 inducibility to a heterologous
promoter in HepG2 cells and in fibroblasts (34).
1 stimulation of COL1A2 promoter in
human mesangial cells and to address the controversy concerning the role of AP-1, Sp1, and Smad proteins in TGF-
1-induced
2(I)
collagen gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 from R&D Systems; goat
polyclonal anti-Smad2/3 IgG (N-19), mouse monoclonal anti-Smad1/2/3 IgG
(H-2), mouse monoclonal anti-Smad4 IgG (B-8), rabbit anti-Sp1 IgG
(PEP-2), normal goat IgG, normal mouse IgG, normal rabbit IgG,
anti-goat IgG-horseradish peroxidase, anti-mouse IgG-horseradish
peroxidase antibody, Sp1 consensus binding site, AP-1 consensus binding
site, Smad3/4 binding site (CAGA), and mutated CAGA oligonucleotide
from Santa Cruz Biotechnology; rabbit anti-phospho-Smad2 IgG from
Upstate Biotechnology Inc.; recombinant protein G-Sepharose and rabbit
polyclonal anti-phosphoserine antibody from Zymed
Laboratories Inc.; anti-rabbit IgG-horseradish peroxidase,
luciferase, and
-galactosidase assay systems from Promega;
Pwo DNA polymerase and rapid DNA ligation kit from Roche Molecular Biochemicals; T4 polynucleotide kinase from Life
Technologies, Inc.; mithramycin A and curcumin from Sigma.
1 or control vehicle for 24 h. These conditions are similar to the ones showing efficient inhibition of Sp1 binding by
mithramycin A and of AP-1 activation by curcumin (32). Total RNA was
analyzed by Northern blot as described previously (36). Autoradiograms
were scanned with an Arcus II Scanner (AGFA) in transparency mode, and
densitometric analysis was performed using the NIH Image 1.61 program
for Macintosh. The same blots were successively rehybridized with
additional probes after confirming complete stripping. cDNAs for
human
1(I) (clone Hf677; Ref. 37) and
2(I) collagen (clone
Hf1131; Ref. 38) chains were obtained from Dr. Y. Yamada (National
Institutes of Health, Bethesda, MD). The cDNA for the human tissue
inhibitor of metalloproteinase (TIMP)-1 (39) was obtained from Dr. D. Carmichael (Synergen, Boulder, CO). The signals obtained by
hybridization with these probes were corrected for loading using the
signal obtained with a bovine cDNA for 28 S ribosomal RNA provided
by Dr. H. Sage (University of Washington, Seattle, WA).
-galactosidase (Life Technologies, Inc.) as a
control of transfection efficiency. Transfection was performed with the
Fugene6 transfection reagent (Roche Molecular Biochemicals) according
to the manufacturer's instructions. After 3 h, 1 ng/ml TGF-
1
or control vehicle was added to the cells. In some experiments, the
transfected cells were pretreated for 2 h with mithramycin A
before adding TGF-
1. Twenty-four hours later, the cells were
harvested in 300 µl of reporter lysis buffer (Promega). Luciferase
and
-galactosidase activities were measured as described previously
(29). Luciferase assay results were normalized for
-galactosidase
activity. Experimental points were realized in triplicate in at least
two independent transfections.
2(I) collagen (COL1A2) promoter and 58 bp
of the transcribed sequence fused to the luciferase (LUC)
reporter gene was described previously (29). The 5' deletion constructs
were generated by PCR amplification using the plasmid pMS-3.5/CAT (40)
as a template and the following pairs of primers:
5'-GCGGATCCATGCAGACAACGAGTCAC-3' or 5'-CAGGATCCGAGTCAGAGTTTCCCC-3' with
3'-CCTCCATGACCGGTGCTCGAGTA-5'. The PCR reactions were carried with the
proofreading Pwo DNA polymerase. After digestion by
BamHI and SstI, the PCR products were extracted
from an agarose gel with the QIAquick gel extraction kit (Qiagen). The
purified fragments were subcloned into the pXP2 vector (41), which
carries the luciferase reporter gene without a promoter. The resulting
constructs were called 268COL1A2-LUC (region from
268 to +50) and
258COL1A2-LUC (region from
258 to +50). Point mutations were
introduced into the potential Smad recognition site (at
268/
260) of
the 376COL1A2-LUC construct using the QuickChange site-directed
mutagenesis kit (Stratagene), according to the manufacturer's
instructions with the following modification: 1 cycle of 1 min at
94 °C; 30 cycles of 1 min at 94 °C, 1 min at 52 °C, 7 min at
72 °C; 1 cycle of 15 min at 72 °C. The primers used were
5'-AGGGCGGAGGTATGTATACAACGAGTCAGAG-3' and
5'-CTCTGACTCGTTGTATACATACCTCCGCCCT-3'. The resulting clone was called
376mCACA-LUC. Mutation and deletion constructs were verified by
sequencing. The vectors expressing the indicated Smad3 variants (42)
were kindly provided by Drs. H. F. Lodish and X. Liu. The
expression vector for Sp1 (43) was kindly provided by Dr. T. Shenk. The
Gal4-Smad constructs (28) were kindly provided by Dr. M. P. de
Caestecker. The Gal4-Sp1 constructs (44) were kindly provided by Dr. R. Tjian.
1-treated cells as described by Schreiber et al.
(45). Double-stranded oligonucleotides were labeled with
[
-32P]ATP and T4 polynucleotide kinase. Five µg of
nuclear extracts were incubated for 30 min at 4 °C with 40,000 cpm
of 32P probe in 10 mM Hepes, pH 7.9, 5%
glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and
1 µg of poly(dI-dC)·poly(dI-dC). For competition and supershift
assays, nuclear extracts were preincubated with a 100-fold molar excess
of cold oligonucleotide or 1 µg of antibody for 30 min or 1 h
before addition of the labeled probe. The DNA-protein complexes were
separated on a 5% polyacrylamide gel in 0.5× TBE buffer and
visualized by autoradiography.
1 for different time periods leading up to simultaneous harvest.
The cells were lysed at 4 °C in lysis buffer (10 mM
Tris/HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) containing
protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin) and phosphatase inhibitors (1 mM sodium
orthovanadate, 50 mM sodium fluoride, 40 mM
-glycerophosphate). Lysates were clarified by centrifugation at
18,000 × g for 10 min. The protein content was
determined by Bradford protein assay (Bio-Rad). Three milligrams of
lysate protein were immunoprecipitated overnight at 4 °C with 6 µg
of anti-Smad2/3 antibody (N-19) or anti-Sp1, followed by precipitation
with 100 µl of protein G-Sepharose for 90 min at 4 °C. After four
washes with complete lysis buffer, the immunoprecipitates were eluted
by boiling for 5 min in 60 µl of 2× Laemmli sample buffer. The
resulting immunoprecipitates were electrophoresed through a 6% or 10%
SDS-polyacrylamide gel, transferred onto a polyvinylidene difluoride
membrane, and immunoblotted with anti-Smad1/2/3, anti-Smad2/3,
anti-Smad4, anti-Sp1, or anti-phosphoSmad2 antibody (0.2 µg/ml), or
anti-phosphoserine antibody (1 µg/ml). The blots were developed with
chemiluminescence reagents according to the manufacturer's protocol
(Santa Cruz Biotechnology). Quantification of the bands on
autoradiograms was performed using densitometric analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-induced Type I Collagen
Expression--
The 5'-flanking region of the human COL1A2
gene contains several GC boxes (Sp1 binding sites) and an AP-1
recognition sequence between nucleotides
340 and
230 (Fig.
1A). This region has been shown to mediate TGF-
responses in fibroblasts, although
contradictory data exist concerning the role of these sites for basal
activity and TGF-
inducibility of the
2(I) collagen promoter
(30-32, 46). Moreover, a CAGA box, which can interact with recombinant
Smad3 and Smad4, is present between the Sp1 and AP-1 binding sites, at
the location
268/
260. Here, we investigated the role of these transcription factors in mediating TGF-
1-induced type I collagen expression in human mesangial cells. Cells were pretreated with mithramycin A, an inhibitor of Sp1 binding (47), or with curcumin, an
inhibitor of c-Jun N-terminal kinase pathway and AP-1 activation (48,
49), before adding TGF-
1 for 24 h. Type I collagen mRNA expression was analyzed by Northern blot. Mithramycin A inhibited both
basal and TGF-
1-induced
1(I) and
2(I) collagen mRNA
levels, whereas curcumin did not significantly affect type I collagen expression (Fig. 1B). These results are consistent with
previous findings from other groups using human fibroblasts (32, 50). The inhibitory effect of mithramycin A was specific since transcription of other genes such as TIMP-1 was not blocked by this chemical antagonist (Fig. 1B). These data suggest that Sp1, but not
AP-1, is involved in induction of type I collagen transcription by
TGF-
1.
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Fig. 1.
Sp1 binding is necessary for
2(I) collagen mRNA expression.
A, representation of the region between
330 and
140 of
COL1A2 promoter. The Sp1 binding sites and AP-1 sequence are
underlined. The CAGA motif is boxed, and a
degenerated CAGA box is indicated by a dotted
line. B, total RNA from cells preincubated with
mithramycin A (100 mM) for 17 h or curcumin (20 µM) for 30 min before addition of 1 ng/ml TGF-
1 or
control vehicle for 24 h was subjected to denaturing
electrophoresis before transfer to a nylon membrane. The same blot was
stripped and reprobed for expression of the indicated mRNA.
cDNA for 28 S rRNA was used as a control for loading. Results from
a representative experiment out of three independent experiments are
shown.
1-responsive
region of COL1A2 in mesangial cells, we performed transient transfection experiments with a series of 5'-deletion mutants of the
2(I) collagen promoter. The construct 376COL1A2-LUC, containing the
sequence from
376 to +58 of the human
2(I) collagen promoter in
front of the luciferase reporter gene, was stimulated about 2-fold by
TGF-
1 (Fig. 2). Deletion of the
sequence between
376 and
268, containing three GC boxes, blocked
the TGF-
1 stimulation of COL1A2 promoter activity.
However, basal expression was not affected. Further deletion, removing
the CAGA box but leaving the AP-1 motif intact, slightly decreased
basal activity and still inhibited TGF-
1 inducibility. These data
indicate that the three GC boxes are necessary for TGF-
1-induced
2(I) collagen promoter activity while the CAGA motif in the context
of the promoter is not sufficient to support ligand stimulation in
mesangial cells. They also confirm that the AP-1 site is not involved
in the activation of the
2(I) collagen gene by TGF-
1.
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Fig. 2.
Identification of a
TGF- 1-responsive region in COL1A2
promoter. Human mesangial cells were transfected with 0.5 µg of the 376COL1A2-LUC construct (containing 376 bp of the
2(I)
collagen promoter fused to the luciferase reporter gene) or the 5'
deletion mutants, 268COL1A2-LUC or 258COL1A2-LUC. The cells were
cotransfected with 0.5 µg of CMV-
-galactosidase as a control of
transfection efficiency. After 3 h, TGF-
1 was added to the
cells at a final concentration of 1 ng/ml. The cells were harvested
after 24 h, and luciferase and
-galactosidase activities were
measured as described under "Experimental Procedures." Luciferase
activity was normalized to
-galactosidase activity. The results are
shown as the mean ± S.E. of transfection experiments performed in
triplicate in five independent experiments.
1 Induction of
COL1A2--
The contribution of Smad3 to TGF-
1-induced
COL1A2 promoter activity was examined. As shown in Fig.
3A, the 376COL1A2-LUC construct was stimulated by TGF-
1 when mesangial cells were
cotransfected with the empty expression vector, pEXL (2.07 ± 0.22-fold induction, n
7). Overexpression of Smad3
increased both basal (11.09 ± 2.82-fold, n
7)
and TGF-
1-induced luciferase activity (a further 3.90 ± 0.47-fold, n
7). In contrast, a Smad3 dominant
negative mutant construct in which the three C-terminal serine residues are replaced by three alanines (Flag-N-Smad3A; Ref. 42) completely inhibited the effect of TGF-
1 (1.08 ± 0.08-fold induction,
n
7). These results are similar to our previous
findings in transient transfection experiments in the presence of serum
(29). However, addition of TGF-
1 to cells cotransfected with Smad3
in complete culture medium led only to a modest induction over
untreated cells (1.44 ± 0.23, n = 4, compared
with 3.90 ± 0.47-fold induction, n = 11, in low
serum-containing medium). The effect of Smad3 overexpression on
COL1A2 promoter activity increased in
dose-dependent manner (Fig. 3B). To further
examine the role of Smad proteins in COL1A2 gene
transcription, mutations were introduced into the CAGA box at
268/
260. These substitutions almost completely blocked TGF-
1 induction of COL1A2 (1.30 ± 0.12-fold induction for
376mCACA-LUC compared with 2.14 ± 0.28-fold induction for
376COL1A2-LUC, n = 3; see Fig. 3C). Together
these data show that overexpressed Smad3 is able to stimulate
2(I)
collagen gene expression in ligand-independent fashion and confirm the
importance of Smad3 in TGF-
1-induced COL1A2 promoter
activity.
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Fig. 3.
Smad3 is involved in
TGF- 1-induced
2(I)
collagen promoter activity. A, cells were cotransfected
with 0.5 µg of the 376COL1A2-LUC construct, and either 0.5 µg of
the vector encoding wild-type Smad3 (Flag-N-Smad3), mutated Smad3
(Flag-N-Smad3A), or the empty expression vector (pEXL), along with 0.5 µg of CMV-
-galactosidase vector. The transfected cells were
treated with 1 ng/ml TGF-
1 for 24 h. Luciferase activity was
normalized to
-galactosidase activity. The results from at least
seven independent experiments are expressed as -fold induction over
untreated cells. B, cells were transfected with 0.5 µg of
the 376COL1A2-LUC and the indicated amount of Flag-N-Smad3. The total
amount of DNA was maintained constant by cotransfection of empty
expression vector. Relative activity of untreated cells cotransfected
with the empty expression vector was arbitrarily set at 1. Relative
luciferase activity is shown on top of each bar,
and -fold induction is indicated on top of each
pair of bars. Results are shown as
relative luciferase activity from at least two independent experiments.
C, cells were transfected with 0.5 µg of either
376COL1A2-LUC or 376mCAGA-LUC. The results from three transfection
experiments are shown as -fold induction.
2(I) collagen promoter activity. Cells were transfected with the
376COL1A2-LUC construct and the Smad3 expression vector. Then, they
were incubated with mithramycin A for 2 h prior to treatment with
TGF-
1. Mithramycin A slightly decreased basal and TGF-
1-induced
376COL1A2-LUC activity in cells cotransfected with an empty expression
vector (Fig. 4). However, it
significantly inhibited basal and ligand-induced effects of Smad3 on
type I collagen promoter activity. In the presence of mithramycin A, COL1A2 promoter activity induced by overexpression of Smad3
was reduced to levels similar to basal COL1A2 promoter
activity, suggesting that ligand-independent stimulation of
2(I)
collagen expression by Smad3 requires Sp1 binding.
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Fig. 4.
Sp1 binding is required for COL1A2
promoter induction by Smad3. Mesangial cells were
cotransfected with 0.5 µg of the 376COL1A2-LUC construct, and either
0.5 µg of the expression vector for wild-type Smad3 or the empty
expression vector, along with 0.5 µg of CMV- -galactosidase vector.
After 3 h, the cells were pretreated with 100 mM
mithramycin A (Mithr.) for 2 h prior to addition of 1 ng/ml TGF-
1. Twenty-four hours later, luciferase and
-galactosidase activities were measured. Luciferase activity was
normalized to
-galactosidase activity. The results are shown as the
mean ± S.E. of transfection experiments performed in triplicate
in three independent experiments.
1 inducibility, an effect that could not be completely overcome
by coexpressing Smad3. Point mutations of the CAGA box, leaving the Sp1
sites intact (376mCAGA-LUC), also inhibited the TGF-
1 response.
However, overexpression of Smad3 increased promoter activity of this
construct. Addition of TGF-
1 further enhanced luciferase activity,
although this induction was not as strong as for the wild-type
construct. The Sp1 binding sites lying between
376 and
268 are thus
necessary for Smad3 to induce COL1A2 activity in a
ligand-independent fashion. Taken together, these results suggest
cooperation between Smad3 and Sp1 in the TGF-
1 response.
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Fig. 5.
Deletion of the three Sp1 sites between
376/
268 decreases the effect of overexpressed Smad3. Cells
were cotransfected with 0.5 µg of the indicated reporter construct,
0.5 µg of Flag-N-Smad3 or pEXL, and 0.5 µg of
CMV-
-galactosidase. Luciferase and
-galactosidase activities were
measured 24 h after addition of TGF-
1. The data represent the
means of a least three independent experiments. The inset
shows the TGF-
1 stimulation for each reporter construct in cells
cotransfected with the empty expression vector pEXL.
1-treated mesangial cells and a
fragment of DNA spanning the COL1A2 promoter sequences from
283 to
250 (WT). This region contains a GC and a CAGA box, and an
AP-1 site. Four DNA/protein complexes were detected (Fig.
6A, complexes
a-d), and their intensity slightly increased with 10 min of
TGF-
1 treatment (compare lanes 2 and
3). This increase was also observed with nuclear extracts
from cells treated with TGF-
1 for 30 or 90 min (data not shown).
Competition experiments with unlabeled probe markedly decreased the
intensity of all four complexes (lanes 4 and
5). Point mutations of the Sp1 site (Mut) abolished its
ability to compete with complexes a and b but not c and d
(lanes 6 and 7), whereas an
oligonucleotide containing a Sp1 consensus binding site was only able
to compete with complexes c and d (lanes 8 and
9). To further demonstrate the presence of Sp1 in complexes
c and d, nuclear extracts were preincubated with anti-Sp1 antibody
(Fig. 6B). In these conditions, a slower migrating complex
was observed while formation of both complexes c and d was partially
inhibited (lanes 4 and 5). In
contrast, normal rabbit IgG did not affect formation of any of these
complexes (lanes 6 and 7). Thus, Sp1
binds to
283/
250 of the COL1A2 promoter and this binding
does not seem to be affected by TGF-
1 treatment. To determine
whether Smad proteins interact with this region, competition
experiments were performed with unlabeled CAGA oligonucleotide, containing three copies of a CAGA box from the PAI1
promoter. When nuclear extracts were preincubated with unlabeled CAGA,
the formation of complex a was prevented, while the intensity of
complexes b-d in TGF-
1-treated cells was decreased (Fig.
6A, lanes 10 and 11). An
AP-1 consensus binding element was unable to compete with the four
complexes. Since the CAGA oligonucleotide used in the competition
experiments was shown to specifically interact with recombinant Smad3
and Smad4 (5), these data suggest that Smad proteins are present in the
complexes with sequences between
283/
250 of COL1A2. When
EMSA were performed with a labeled COL1A2 oligonucleotide containing mutations of the Sp1 site (Mut), two shifted complexes were
observed (Fig. 6C, lanes 2 and
3). These complexes correspond to the two fastest migrating
bands (a and b) obtained with the wild-type COL1A2 sequence.
Both complexes were competed by incubation with unlabeled
oligonucleotide (lanes 4 and 5) or
unlabeled CAGA consensus binding site (lanes 6 and 7). We were unable to detect the presence of Smad
proteins using a specific antibody (see "Discussion"). Nevertheless, the competition experiments with a Smad consensus binding
site and data from Chen et al. (33) suggest that Smad proteins interact with the COL1A2 promoter. Taken together,
these results indicate formation of nucleoprotein complexes between
283/
250 COL1A2 and Sp1 and Smad proteins.
View larger version (55K):
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Fig. 6.
Sp1 and Smad binding to the COL1A2
promoter. A, nuclear extracts from control cells
( ) or cells treated with 1 ng/ml TGF-
1 for 10 min (+) were
incubated with a labeled oligonucleotide spanning COL1A2
promoter sequences between
283 and
250 (WT). For competition
experiments, extracts were preincubated for 30 min with a 100-fold
molar excess of the indicated unlabeled oligonucleotides. B,
supershift experiments were performed by preincubating the extracts
with antibody for 1 h before addition of labeled WT
oligonucleotide. An arrow indicates the supershifted
complex. C, nuclear extracts were incubated with labeled
COL1A2 oligonucleotide mutated at the Sp1 site
(Mut) or WT oligonucleotide. Unlabeled oligonucleotides used
in competition experiments are indicated.
1 Increases Association between Sp1 and Smad
Proteins--
We next examined whether members of the Smad family can
form complexes with Sp1 in human mesangial cells. Lysates from cells treated with TGF-
1 for the indicated periods of time were
immunoprecipitated with either an anti-Smad2/3 (Fig.
7) or anti-Sp1 (Fig.
8) antibody. The coimmunoprecipitated
proteins were analyzed by immunoblotting blot with the indicated
antibodies. As shown in Fig. 7, TGF-
1 increased association of
Smad2/3 with Sp1 within 10 min of treatment. The increased interaction
between Smads and Sp1 was maximal between 10 and 90 min of treatment,
and this interaction remained higher than in control cells for up to
24 h (Fig. 7 and data not shown). Consistent with our previous
observation, the association between Smad2/3 and Smad4 also increased
and followed the pattern of Smad2/3 phosphorylation, starting within 5 min after adding TGF-
1. The phosphorylation and association peaked
at 15-30 min and decreased by 90 min, but remained higher than
baseline for up to 24 h (Fig. 7; Ref. 29 and data not shown). When
Sp1 was immunoprecipitated, a similar pattern and timing of association
with Smad proteins was observed (Fig. 8). These effects were not due to
a change in protein levels as shown by Western blot analysis on whole
cell lysates. Of note, only the 105-kDa form of Sp1 was detected in the
Smad3 immunoprecipitates (Fig. 7) while Smad3, but not Smad2, was
present in Sp1 immunoprecipitates (Fig. 8). These results indicate that
TGF-
1 induces Sp1 and Smad protein interaction. However, this
association begins before maximal Smad3 phosphorylation occurs.
View larger version (49K):
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Fig. 7.
Coimmunoprecipitation of Sp1 and Smad4 with
Smad2/3. Cells were treated with 1 ng/ml TGF- 1 for the
indicated periods of time leading up to simultaneous harvest. After
immunoprecipitation (IP) using anti-Smad2/3 antibody (N-19),
the resulting complexes were electrophoresed through polyacrylamide
gels followed by immunoblotting. The blots were developed with the
indicated antibodies. Whole cell lysates were analyzed in parallel.
A, the left panels show a
representative coimmunoprecipitation experiment. No complexes were
detected in lysates immunoprecipitated with normal goat IgG (data not
shown). Western blot analysis on whole cell lysates from the same
experiment is shown in the right panels.
B, results from densitometric analysis of five independent
experiments. The data are shown as the ratio between
coimmunoprecipitated Sp1 (left) or Smad4 (right)
and immunoprecipitated Smad2/3.
View larger version (35K):
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Fig. 8.
TGF- 1 increases
association between Sp1 and Smad3 and Smad4. Mesangial cells were
treated and analyzed as described in Fig. 7, except that an anti-Sp1
antibody (PEP-2) was used for immunoprecipitation (IP).
A, the left panels show a
representative coimmunoprecipitation experiment. No complexes were
detected in lysates immunoprecipitated with normal rabbit IgG (data not
shown). Western blot analysis on whole cell lysates from the same
experiment is shown in the right panels.
B, results from densitometric analysis of least three
independent experiments. The data are shown as the ratio between
coimmunoprecipited Smad3 (left) or Smad4 (right)
and immunoprecipitated Sp1.
1--
The above experiments indicate that endogenous Smads
and Sp1 can form complexes in human mesangial cells. We then
investigated whether their interaction can lead to transcriptional
activation using the Gal4-LUC assay system. A reporter construct
containing five Gal4 binding sites in front of the luciferase gene
(pFR-LUC) was cotransfected with the Gal4 DNA binding domain fused to
full-length Smad3 (Gal4-Smad3) or Smad4 (266) (Gal4-Smad4(
N)),
and with a vector expressing Sp1 or the control empty vector. TGF-
1
stimulated transcriptional activity of Gal4-Smad3, Gal4-Smad4(
N), or
both by 1.75-, 4.29-, and 2.66-fold, respectively (Fig.
9A). Overexpressed Sp1
enhanced Smad3-mediated transcription (2.27-fold increase over activity
in control cells transfected with empty expression vector). This effect
was further increased after treatment with TGF-
1. In contrast to
Smad3, the basal activity of Gal4-Smad4 (266) was not increased by
Sp1. However, overexpressed Sp1 enhanced responses to TGF-
1.
Ligand-independent and ligand-dependent stimulation by Sp1
was markedly increased when cells were cotransfected with a combination
of Gal4-Smad3 and Gal4-Smad4(
N). Neither TGF-
1 nor Sp1 was able
to activate the reporter construct cotransfected with the Gal4 DNA
binding domain alone. Thus, the interaction between Sp1 and Smad3
observed in Figs. 7 and 8 correlates with the functional cooperation
between those proteins. Moreover, the TGF-
1-increased association
leads to increased transcriptional cooperativity between Smads and
Sp1.
View larger version (17K):
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Fig. 9.
Functional interaction between Sp1 and Smad
proteins. Cells were transfected with 0.5 µg of the pFR-LUC
reporter construct (containing five Gal4 binding sites in front of the
luciferase gene), 0.5 µg of the indicated Gal4 DNA binding domain
fusion proteins (or 0.25 µg in double transfection), and 0.5 µg of
CMV- -galactosidase as a control of transfection efficiency. Cells
were cotransfected with an expression vector for Sp1 or the control
vector (A) or with an expression vector for Smad3 or the
control vector (B). Twenty-four hours after treatment with
TGF-
1, luciferase and
-galactosidase activities were measured.
Luciferase activity was normalized to
-galactosidase activity.
Results are shown as the mean ± S.E. of triplicate wells from a
representative experiments. Similar results were obtained in three
independent experiments.
1 had no effect,
suggesting that, in our system, this factor does not directly stimulate
transcriptional activity of the B domain of Sp1. Cotransfected Smad3
enhanced transcription by Gal4-Sp1(B) and Gal4-Sp1(B-c) only in the
presence of TGF-
1 (~2-fold increase over untreated cells), while
the phosphorylation-deficient Smad3 mutant had no effect (data not
shown). Only low levels of reporter activity were detected with the
Gal4 DNA binding domain alone or Gal4-Sp1(B-n), and these levels were
not affected by TGF-
1 or Smad3. These findings further support a
functional cooperation between Sp1 and Smad3 to induce transcription in
a ligand-dependent manner, and suggest that Sp1 enhances
TGF-
1-stimulated Smad transcriptional activity.
2(I) Collagen Gene
Expression--
We have shown that Sp1 and Smad3 can form complexes in
human mesangial cells and that these proteins functionally interact in
a Gal4 transactivation assay system. To evaluate whether this interaction is involved in
2(I) collagen gene expression, mesangial cells were cotransfected with the TGF-
1-responsive collagen
construct, 376COL1A2-LUC, or with 5' deletion mutants and expression
vectors for Smad3, Sp1, both, or the corresponding empty vectors.
TGF-
1 increased 376COL1A2-LUC activity by 2-3-fold in cells
cotransfected with the empty expression vectors, whereas it did not
stimulate the shorter COL1A2-LUC constructs (Fig.
10A). Overexpression of Sp1
did not affect basal or ligand-induced expression of 376COL1A2-LUC. Sp1
also had no effect on 268COL1A2-LUC or 258COL1A2-LUC activity. On the
other hand, Smad3 increased 376COL1A2-LUC activity in the absence or
presence of TGF-
1 by 11.09 ± 2.82- and 16.32 ± 1.68-fold, respectively, over cells transfected with empty expression
vector (n = 12). Similar to results presented in Fig.
5, overexpressed Smad3 had a limited effect on TGF-
1-induced
luciferase activity of the 5' deletion constructs. These results
suggest that Sp1 is saturated for expression of the
2(I) collagen
promoter in mesangial cells, while Smad3 is the limiting factor.
Indeed, when Sp1 and Smad3 were overexpressed together, they induced
marked ligand-independent and ligand-dependent
transcriptional activity of 376COL1A2-LUC (Fig. 10B). When
sequences between
376 and
268, containing three Sp1 sites, or
between
376 and
258, containing three Sp1 sites and one CAGA box,
were deleted, responsiveness to TGF-
1 could be rescued only
partially by coexpressing Smad3 and Sp1 together (Fig. 10A).
These results suggest that, when Smad3 and Sp1 are overexpressed, they
could form complexes able to bind to more proximal regulatory regions
of less affinity, while endogenous levels of these proteins would not
be able to interact with these sequences to stimulate transcription in
response to TGF-
1.
View larger version (23K):
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Fig. 10.
Sp1 and Smad3 cooperate to stimulate
TGF- 1-induced
2(I)
collagen promoter activity. A, mesangial cells were
transfected with 0.5 µg of the indicated COL1A2-LUC reporter
construct and 0.5 µg of expression vector for Sp1, 0.5 µg of
expression vector for Smad3, or 0.5 µg of each expression vector. The
total amount of DNA was maintained constant by cotransfection of empty
vectors. The cells were also cotransfected with 0.5 µg of
CMV-
-galactosidase. After incubation with TGF-
1, luciferase and
-galactosidase activities were measured. Luciferase activity was
normalized to
-galactosidase activity. Experimental points were
realized in triplicates in two independent transfections. Values are
given as mean ± S.E. of a representative experiment. The
inset shows the TGF-
1 stimulation for each reporter
construct in cells cotransfected with the empty expression vector or
the expression vector for Sp1. B, cells were transfected 0.5 µg of 376COL1A2-LUC, 0.5 µg of CMV-
-galactosidase, and the
indicated amount of expression vector.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2(1) collagen gene expression in
response to TGF-
1 in glomerular mesangial cells. While this manuscript was in revision, several manuscripts from other groups, which have been published or accepted for publication, showed cooperation between Sp1 and Smad proteins in mediating
p15Ink4B,
5 integrin subunit,
COL1A2, and PAI-1 gene expression (51-54). Our
data agree with these findings and extend them by showing direct
evidence of interaction among endogenous Sp1 and Smad3 and Smad4 in
nontransformed cells. In addition, our experiments show that this
interaction leads to ligand-induced activation and cooperation of these
proteins. These results provide strong support for the concept that
this mechanism contributes to pathophysiological collagen accumulation
in human renal disease, which has been associated with increased
TGF-
1 activity.
376 and
265, encompassing three Sp1 consensus sites, decreased basal
COL1A2 expression (31, 46). However, one study showed that
deletion of the two upstream Sp1 sites reduced basal promoter activity
by about 50%, while deletion of the third site had no effect. The
other study showed that the deletion of all three sites was necessary
to alter the promoter expression. Our data indicate that the region
between
376 and
265 does not play a role in basal expression of
COL1A2 in mesangial cells. Mithramycin A, an inhibitor of
Sp1 binding, decreased basal
1(1) and
2(1) collagen mRNA
expression by ~50% (Fig. 1B). In contrast, in transient transfection experiments, this biochemical antagonist only slightly inhibited basal activity of the construct containing the promoter regulatory sequences from
376 to +58 (Fig. 4). These results suggest
that further upstream and/or downstream regions are involved in basal
COL1A2 gene transcription. Indeed, a positive regulatory region for fibroblast expression has been identified between
3500 and
772 (31, 40). Ihn and collaborators (55) have also identified
positive cis-elements at
128/
123 and
84/
80.
Mutations of either site led to ~90% decrease in basal promoter
activity in fibroblasts. The Sp family members, Sp1 and Sp3, were shown to bind to the TCCTCC motif at
128/
123 (56). These regions might
also play a role in mesangial cell COL1A2 gene expression.
1-induced
2(1) collagen gene
transcription in human mesangial cells. We have defined a
TGF-
1-responsive element in the COL1A2 promoter that
contains three Sp1 binding sites and is located between
376 and
268
upstream from the initiation site (Fig. 2). A similar region, lying
between
330 and
255, confers TGF-
responsiveness to a
heterologous promoter in human dermal fibroblasts (30). In the same
study, the authors also showed that optimal response to TGF-
depends
on the structural integrity of the Sp1 sites. In contrast, Chung
et al. (31) showed that deletion of the sequence between
376 and
265 did not affect TGF-
1-induced COL1A2
promoter activity in fibroblasts while the region between
265 and
241, containing an AP-1 site, was essential for ligand induction.
There is no obvious explanation for these discrepancies in fibroblasts
except the experimental conditions and/or the TGF-
isoforms used.
However, our data exclude that AP-1 binding is required for the
TGF-
1 stimulation of COL1A2 in human mesangial cells, in
agreement with data obtained in fibroblasts by Greenwel et
al. (32). Mutation of the CAGA box (at
268/
260) decreased
TGF-
1 responses in mesangial cells (Fig. 3C) as well as
in fibroblasts. Six tandem repeats of this sequence are sufficient to
induce TGF-
1 responsiveness to a minimal promoter (30, 34). However,
our deletion experiments also indicate that, without the upstream Sp1
binding sites, the CAGA box is not able to confer TGF-
1
inducibility. This suggests that, in the context of the COL1A2 promoter, a single CAGA sequence requires the
presence of Sp1 regulatory element(s) to support the TGF-
1 response.
Further support of the importance of Sp1 binding for
TGF-
1/Smad-mediated stimulation of COL1A2 comes from
experiments in which human mesangial cells were cotransfected with 5'
deletion mutants of the COL1A2 promoter and Smad3 expression
vector. In the absence of the Sp1 binding sites located between
376
and
268, overexpressed Smad3 cannot fully stimulates
COL1A2 promoter activity (Fig. 5). In contrast, when the
CAGA box between
268/
260 is mutated, overexpressed Smad3 is still
able to stimulate COL1A2 transcription in a
ligand-independent manner, suggesting that the effect of Smad3
overexpression on promoter activity is mediated through the Sp1 binding
sites. Thus, our results indicate that both Sp1 binding sites and the
CAGA box located between
376 and
258 play a role in
TGF-
1-induced
2(1) collagen promoter activity in human mesangial cells.
1-induced
activity of the 376COL1A2-LUC construct, whereas Smad3 overexpression
enhances basal expression and TGF-
1 responsiveness in a
dose-dependent manner (Figs. 3B and
10A). These data indicate that Smad3 is the limiting factor
for TGF-
1-induced
2(I) collagen expression. When Sp1 and Smad3
are overexpressed together, they cooperate to increased
COL1A2 promoter activity. The low levels of stimulation of
COL1A2 by Sp1 and TGF-
1-activated Smad3 in the absence of
the GC and CAGA sequences between
376 and
258 suggest that, when
overexpressed, Sp1 and Smad3 could bind to more proximal regions of the
promoter. Indeed, the TCCTCC motif at
128/
123, which was found to
constitute a Sp1 binding site and to play a role in basal
COL1A2 expression in fibroblasts (56), is separated by 3 bp
from a motif similar to the Smad3/4 response element described by
Dennler et al. (5). These regions could support TGF-
1
inducibility when Sp1 and Smad3 are overexpressed. This hypothesis is
under investigation.
283 and
250 was demonstrated by gel shift experiments. The presence of Smads in nucleoprotein complexes with the same promoter elements was
also suggested by specific competition experiments. However, we were
unable to detect Smad binding with a specific anti-Smad1/2/3 antibody.
The same antibody recognized the interaction of endogenous Smads with
an oligonucleotide containing three repeats of a CAGA box.2 (53). reported similar
findings in NIH3T3 cells. The failure of the antibody to induce a
supershift of Smad proteins in complex with the COL1A2
promoter could be due to the relatively low amount of Smads in these
nucleoprotein complexes. Indeed, Smad binding to the
TGF-
1-responsive element in the COL1A2 promoter could only be demonstrated in NIH3T3 cells when both Smad3 and Smad4 were
overexpressed (53). Another possible explanation is that Sp1 and/or
other nuclear factors could impair recognition by the anti-Smad
antibody used in these experiments.
1, suggesting that Sp1 and Smad3/4 can interact in
the absence of Smad3 phosphorylation. There is increased interaction
upon ligand stimulation. This effect is maximal between 10 and 90 min
after adding TGF-
1, similar to Smad3 phosphorylation (Figs. 7 and
8). However, the amount of Smad/Sp1 complex detected remains higher
than in control cells for up to 24 h. These results suggest that
this minimal association between Sp1 and Smad3/4 might play a role in
the sustained response to TGF-
1. In the coimmunoprecipitation
experiments, Smad3 and Smad4, but not Smad2, were detected in Sp1
immunoprecipitates, suggesting that, in human mesangial cells, Sp1
preferentially associates with endogenous Smad3 and Smad4. However, we
could not completely exclude association with Smad2, since the antibody
used for detection of Smad2 and Smad3 recognizes Smad2 with less
affinity. Moreover, in the Gal4-LUC assay system, TGF-
1-induced
Gal4-Smad2 transcriptional activity is increased in the presence of
Sp1,2 indicating that when overexpressed these two proteins
can interact. Pardali et al. (57) have shown, by glutathione
S-transferase pull-down analysis, that Smad2, Smad3, and
Smad4 can directly interact with Sp1. Coimmunoprecipitation of tagged
proteins transiently expressed in COS-7 cells showed that there is
constitutive association between Sp1 and Smad3 and a weaker association
with Smad2. Activation of the TGF-
signaling pathway by means of a
constitutively active type I receptor increased these interactions.
These data are similar to our results obtained with endogenous Smad and
Sp1 proteins from human mesangial cells. Of note, only the 105-kDa form
of Sp1 is detected in coimmunoprecipation with Smad3. Since this species results from phosphorylation of the 95-kDa form and this modification occurs in vitro only when DNA is present in the
reaction (58), this would argue that association of endogenous Sp1 and Smad3 occurs when Sp1 is bound to DNA.
1.
The transcriptional cooperation between Sp1 and Gal4-Smad3 or
Gal4-Smad4(
N) is further increased when both chimeric proteins are
expressed together, suggesting that Sp1, Smad3, and Smad4 synergistically interact to stimulate transcription. Using the same
Gal4-LUC assay system with Gal4-Sp1 chimeric proteins, we showed that
the C-terminal part of the B domain of Sp1 functionally cooperates with
ligand-activated Smad3 to stimulate gene transcription (Fig.
9B). The B domain of Sp1 is able to activate transcription from a heterologous Gal4 promoter independently of TGF-
1
stimulation. Thus, in our system, the transcriptional activity of the B
domain of Sp1 is not regulated by TGF-
1. In contrast, Li et
al. and Feng et al. (51, 59) showed that TGF-
1 could
stimulate the transactivating activity of the B domain of Sp1 in HaCaT
cells. These differences might reflect cell-specific responses to
TGF-
1. TGF-
1 could regulate Sp1 activity by modulating the
activity of Sp1-associated proteins that are not present or are present in too low concentration in human mesangial cells compared with HaCaT
cells. Indeed, when Smad3 is coexpressed, the Gal4-Sp1(B) and
Gal4-Sp1(B-c) constructs become responsive to TGF-
1.
1 could also modulate the activity of Sp1 domains other than the
transactivating domain B. In fact, Sp1 DNA binding activity has been
shown to be modulated by several kinases including extracellular signal-regulated kinase 2 and protein kinase A activity (60, 61). Since
we and others have shown that TGF-
1 stimulates extracellular signal-regulated kinase 1/2 and protein kinase A activity in mesangial cells (62, 63), these kinases could contribute to modulation of Sp1
activity in response to TGF-
1. Recently, Moustakas' group showed
that Smad3 increased activity of Gal4-LUC induced by chimeric proteins
of Gal4 linked to various domains of Sp1 in HepG2 cells (57, 64).
However, the effect of TGF-
1 was not examined. Our experiments show
that Smad3 has no significant inducing activity on Gal4-Sp1(B) or
Gal4-Sp1(B-c) in the absence of TGF-
1, perhaps reflecting a
cell-specific response. In summary, our results suggest that
ligand-activated Smad3 either acts as a cofactor to Sp1 transcriptional activity or recruits other coactivators to modulate Sp1 transactivating activity.
1 by interacting together and with TAFs and recruiting
coactivators such as CBP/p300 to the initiation transcription complex.
1. Sp1 and
Smad proteins form complexes, and their synergistic cooperation plays
an important role in mediating TGF-
1-induced
2(I) collagen expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susan C. Hubchak for isolating and
characterizing the human mesangial cells. We appreciate generous
provision of the cDNAs for human 1(I) and
2(I) collagen by
Dr. Y. Yamada, the TIMP-1 cDNA by Dr. D. Carmichael, the 28 S
cDNA by Dr. H. Sage, the Flag-N-Smad3 and Flag-N-Smad3A by Drs.
H. F. Lodish and X. Liu, the expression vector for Sp1 by Dr. T. Shenk, the Gal4-Smad3 and Gal4-Smad4 (266) constructs by Dr.
M. P. de Caestecker, and the Gal4-Sp1(B), Gal4-Sp1(B-c), and
Gal4-Sp1(B-n) constructs by Dr. R. Tjian. We also thank members of the
Schnaper laboratory for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant ROI DK49362 from NIDDK, National Institutes of Health.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.
Supported by a research fellowship from the National Kidney
Foundation. To whom correspondence should be addressed: Northwestern University Medical School, Pediatrics W-140, 303 E. Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-0089; Fax: 312-503-1181; E-mail: anne-c@northwestern.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M006442200
2 A.-C. Poncelet and H. W. Schnaper, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ECM, extracellular
matrix;
TGF-, transforming growth factor-
;
COL1A2,
2(I) collagen gene;
R-Smad, receptor-regulated Smad;
SBE, Smad
binding element;
FBS, fetal bovine serum;
TIMP, tissue inhibitor of
metalloproteinase;
LUC, luciferase;
EMSA, electrophoretic mobility
shift assay;
WT, wild type;
PCR, polymerase chain reaction;
bp, base pair(s).
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REFERENCES |
---|
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