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INTRODUCTION |
TGF-
1,1 a
multifunctional regulatory cytokine, generally stimulates cell
growth and production of extracellular matrix in mesenchymal
cells (1-6). It is produced by chondrocytes and abundantly stored in
cartilage extracellular matrix, where it may contribute to the repair
process in response to a variety of stimuli (7-9). TGF-1 exerts its
action through a multimeric complex involving two membrane
serine/threonine kinase receptors and an intracellular signaling
pathway involving a cascade of Smad proteins (10). Smad2 and Smad3 are
direct substrates of the TGF-1 kinase receptors and interact with
Smad4 (11-13). These Smad complexes then translocate to the
nucleus where they function as transcriptional factors. Smads interact
with various partner proteins and thereby exhibit a wide variety of
biological activities (14). Cooperation with FAST-1 (15), Sp1 (16-19),
AP-1 (20, 21), VDR (22), and p300/CBP co-activator protein (23) has
been implicated in the Smad-mediated transcription of TGF-1
responsive genes. By contrast with receptor-activated Smads, the
inhibitory Smads appear to serve as an autoregulatory negative feedback
function in cellular TGF-1 signaling by competitive interaction with
the type I TGF-1 receptor (24-27).
Other pathways, sometimes in specific contexts, may also be implicated
in TGF-1 signaling in various cell types, such as the
stress-activated protein kinase/c-Jun amino-terminal kinase (28) and
MAP kinases, including extracellular signal-activated kinase and p38
MAP kinase. For example, the p38 MAP kinase plays crucial roles in the
induction by TGF-1 of the chondrogenic phenotype in chondroblastic
cells (29). In chondrogenic ATDC5 cells, TGF-1 induced aggrecan gene
transcription through cross-talk between Smad2, extracellular
signal-activated kinase 1/2, and p38 MAPK pathways (30).
Articular cartilage, a highly specialized connective tissue, consists
of relatively few chondrocytes distributed throughout an abundant
extracellular matrix, including type II, IX, and XI collagens and a
large proteoglycan aggrecan (31). These matrix components are
synthesized by chondrocytes that are responsible for the maintenance of
a regulated balance between the anabolism and the catabolism of the
cartilage-specific macromolecules. Extracellular matrix synthesis is
controlled by several factors such as insulin-like growth factor-1 and
TGF-1, whereas its degradation may be induced by proinflammatory
cytokines like interleukin-1 or tumor necrosis factor-
(32, 33).
Alteration of type II collagen expression, an essential phenotypic
marker of cartilage, is associated with a variety of joint diseases
such as osteoarthritis and rheumatoid arthritis (34-36). In cartilage
degenerative diseases, chondrocytes have been found to de-differentiate
(37, 38). Therefore, it is of special interest to get insight into the
molecular mechanisms that regulate type II procollagen gene
(COL2A1) expression in normal and de-differentiated chondrocytes.
We have previously identified a 458-bp region in the first intron that
mediates enhancer activity and a 266-bp short promoter that is also
responsible for high level expression of the human COL2A1
gene (39). These two regions contain several C-Krox-, Sp1-, and Sp3
DNA-binding sites that modulate the in vitro transcriptional activity of this gene (39, 40). The Sp gene family of transcription factors consists of five members, which are referred to as Sp1-Sp5, that bind with similar affinity to GC-rich motifs (41, 42). Whereas
Sp1, Sp2, and Sp4 are transactivators, Sp3 is generally considered as a
repressor of transcription (41). We found that Sp1 and Sp3 from RAC
nuclear extracts bound to six Sp DNA-binding sites within the 
266-bp
COL2A1 promoter whereby they exert their transcriptional
effects (40). Concomitant overexpression of the two Sp proteins
revealed that Sp3 prevented the Sp1 induction of COL2A1
promoter activity. Sp1 specifically activated type II collagen
neosynthesis while Sp3 inhibited it, suggesting that type II
collagen-specific expression in chondrocytes is likely to depend on the
Sp3/Sp1 ratio (40).
The TGF-1 effect on type II collagen synthesis by chondrocytes has
been found to greatly vary as a function of several experimental factors, so that already reported data may appear contradictory. For
example, we previously demonstrated that TGF-1 was capable of
stimulating type II collagen expression in short-term treatment (24 h),
using confluent serum-deprived monolayers of RAC (43). A similar effect
was also reported for chondrocytes cultured in alginate beads (44, 45).
In contrast, we found that TGF-1 exerted an inhibitory effect on
collagen synthesis in a 6-day exposure of RAC cultures (46). The
TGF-1 action on type II collagen synthesis by chondrocytes may also
depend on whether the cells are fully differentiated or have undergone
phenotype alteration (46).
Although it is now proven that TGF-1 may exert bifunctional
regulation of type II collagen expression in chondrocytes, so far
little information has been gained on the molecular mechanisms underlying the cytokine control of COL2A1 transcription.
Horton et al. (47) demonstrated that TGF-1 and basic
fibroblast growth factor acted in a synergistic fashion to suppress the
synthesis of type II collagen on embryonic chicken sternal chondrocytes through a transcriptional control involving the intronic
chondrocyte-specific enhancer of the COL2A1 gene and
silencer elements present in the promoter region. This mechanism was
found to be dependent on protein kinase C activation (48). In addition,
a previous report has shown that the effect of TGF-1 on type II
collagen expression, in rat articular chondrocytes, could also
implicate activation of extracellular signal-activated kinases and
subsequent AP-1 binding (49).
The preceding data suggest that the TGF-1 effect on type II collagen
production is likely to be mediated through cis-binding sequences of the promoter and/or first intron regions of the
COL2A1 gene so far uncharacterized. Here, we demonstrate for
the first time that TGF-1 down-regulation of COL2A1 gene
transcription implicates a region covering 63-bp upstream of the
transcription start site and that this effect is dependent on the
increase of the Sp3/Sp1 ratio.
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EXPERIMENTAL PROCEDURES |
Cell Cultures--
RAC were prepared from the shoulders and
knees of 3-week old rabbits, as previously described (46, 50). Cells
were seeded at 2 × 104 cells/cm2 in
either 6-well plates, 100-mm dishes, or 75-, 150-, and
175-cm2 flasks and cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal
calf serum (FCS), glutamine (2 mM), penicillin (100 IU/ml),
streptomycin (100 µg/ml), and Fungizone (0,25 µg/ml) in a 5%
CO2 atmosphere. The medium was changed twice a week.
Transfection Experiments--
Chondrocytes seeded at a density
of 2 × 104 cells/cm2 in either 6-well
plates or 100-mm dishes were transiently transfected at 80% confluency
by the calcium phosphate precipitation method (39, 40). The reporter
plasmids (10 µg each) were cotransfected with a pSV40
-galactosidase expression vector (2 µg) as an internal control of
transfection efficiency, and/or with an expression pCMV5 vector (15 µg) with or without the cDNA encoding a hybrid TGF-1 receptor
called TRII/I. 10-15 h after transfection, the medium was changed
and the cells were incubated with or without TGF-1 (1-3 ng/ml) in
DMEM with or without 10% FCS. 24 h later, the samples were
harvested and the protein content, luciferase, and -galactosidase
activities were assayed. Luciferase activity was measured on total cell
extracts (kit from Promega) in a luminometer (Berthold Lumat LB 9501).
-Galactosidase activity was assayed with a colorimetric assay (51),
whereas the protein amount was determined by the Bradford colorimetric
method (Bio-Rad). Luciferase activities were normalized to
transfection efficiency and protein amount and expressed in relative
luciferase units (RLU) as the mean ± S.D. of three independent samples.
In some experiments, reporter plasmids were also cotransfected with Sp1
and/or Sp3 expression vectors (pEVR2/Sp1 and/or pRC/CMV/Sp3, respectively) as previously described (40, 52). The corresponding insertless expression vectors were used as controls (pEVR2 and pRC/CMV). In these experiments, the pSV40-gal plasmid has not been
cotransfected because Sp1 is able to increase SV40 promoter activity,
because of the reported presence of several Sp1 DNA-binding motifs in
this promoter (40). Chondrocytes have also been incubated, in
some experiments, with 100 nM mithramycin (Sigma) for
24 h, following the 10-15 h transfection period, to block Sp1 and
Sp3 interaction to their respective DNA-binding sites.
Decoy Oligonucleotide Assays--
Decoy double-stranded
oligonucleotides were transfected or added to RAC cultures in an
attempt to interfere with Sp1 and Sp3 binding to their cognate
cis-acting elements within the 63-bp short promoter of the
COL2A1 gene. The sequences of the 67/30wt, 67/30mut,
50/+1wt, 35/15wt, 35/15mut, Sp1mcwt, and Sp1mcmut decoy
oligonucleotides are presented in Table I. These oligonucleotides were
cotransfected in the RAC cultures together with the reporter constructs. In certain experiments, the decoy oligonucleotides were not
transfected but simply added to the fresh culture medium with or
without TGF-1 after the overnight transfection period. After an
incubation of 24 h, cell lysates were prepared and assayed for
luciferase, -galactosidase activities, and protein amounts.
DNA Constructions--
Most of the COL2A1-luciferase
reporter vectors have been previously described (39, 40). However, two
additional plasmids have been generated. Thus, the pGL2-0.387kb plasmid
was used to obtain a 82-bp BglII-HindIII DNA
fragment (35/+47 bp of COL2A1 gene) and a 110-bp
BglII-HindIII (63/+47 bp of
COL2A1 gene) insert by PCR, using the following primers:
sense 351(II), 5'-CCGGAGATCTGGCGCATATAACGGGC-3' (BglII site in bold); sense 631(II),
5'-CCGGAGATCTGCGATTCGCCAG-3' (BglII site in
bold); antisense +471(II), 5'-CCGGAAGCTTGGAGCAGGAGGAG-3' (HindIII site in bold). PCR conditions were
95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.
The 82- and 110-bp BglII-HindIII fragments were
then subcloned in the respective sites present in the polylinker of
pGL2-basic giving, respectively, pGL2-0.082kb and pGL2-0.110kb. These
reporter plasmids were sequenced to confirm the accuracy of the constructions.
The reporter plasmids have been also cotransfected with a pCMV5
expression vector containing the cDNA coding for a hybrid TGF-1
receptor called TRII/I, which induces a dominant negative effect on
growth factor signaling (53). To confirm the specificity of TGF-1
effects on COL2A1 gene transcription in our experimental model we used the p3TPLux reporter vector kindly provided by Dr. J. Massagué (New York) (54). To measure the specificity of Sp1 and
Sp3 effects, pGL2mcSp1wt and pGL2mcSp1mut reporter plasmids, previously
described, have been included in the experiments (40).
Nuclear Extracts and DNA Binding Analysis--
Nuclear extracts
were prepared as maxi- or minipreparations (55, 56). EMSAs were
performed with the oligonucleotides shown in Table I, as previously
described (40). In the antibody interference assays, 1 µl of
anti-Sp1, anti-Sp3, anti-p50, anti-p65, anti-Smad3, anti-AP2, and
anti-EGR2 antibodies (Santa Cruz) were added to each reaction mixture
for 15-20 min at room temperature, then the incubation was prolonged
for 15 min at 4 °C. The probe was finally added in the binding
reaction and a further 15-min incubation at room temperature was
performed. Then, the samples were run on a 6% polyacrylamide gel, for
2 h at 150 V, in nondenaturing conditions. For DNase I footprint
experiments, the SmaI-HindIII fragment of the
pGL2-0.110kb plasmid was end-labeled to its 5' SmaI
extremity. Further processing of the probe including gel purification
and elution was performed as previously described (39, 40). For
Southwestern analysis, 40 µg of nuclear extracts were run on a 10%
polyacrylamide electrophoresis gel in denaturing conditions. After
electrotransfer, the polyvinylidene difluoride nylon membrane was
processed according to the method of Singh et al. (57),
including a denaturation-renaturation protocol, using the
+2440/+24851(II) wild-type and mutant oligonucleotides as probes
(40). For quantification, the autoradiogram was scanned using a
StudioScan II SI scanner (AGFA). Then, the intensity of the
corresponding bands was quantified by densitometric scanning using
ImageQuant software (Molecular Dynamics).
Reverse Transcriptase-PCR Analysis--
Total RNA was extracted
as previously reported (40) and 2 µg of total RNA were reverse
transcribed into cDNA in the presence of 50 pmol of oligo(dT), 40 units of RNAseOut (Invitrogen), 10 mM of each dNTPs
(Invitrogen), first-strand buffer 5×, and 60 units of Moloney murine
leukemia reverse transcriptase (Invitrogen). The reaction was performed
at 42 °C for 15 min, and followed by a 5-min step at 99 °C and a
5-min step at 4 °C, using an Omni-E-Hybaid thermocycler and a
PCR kit (Invitrogen). The following primers were used:
COL2A1, sense, 5'-GACCCCATGCAGTACATG-3';
antisense, 5'-GACGGTCTTGCCCCACTT-3' (58); Sp1, sense,
5'-CTACCCCTACCTCAAAGG-3'; antisense, 5'-CTCTCCTTCTTTTTGCTGG-3'; Sp3,
sense, 5'-TAAGGTGTATTGCGTCTT-3'; antisense, 5'-TGAGGTGGTCTTAAGAAT-3'
(59); and GAPDH, sense, 5'-TGGTATCGTGGAAGGACTCATGAC-3'; antisense,
5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' (60).
A variable number of PCR cycles were done as follows: 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. cDNAs were
analyzed by 2% agarose gel electrophoresis and visualized by ethidium
bromide staining. The amplification reaction yielded the expected
cDNA sizes (COL2A1, 648 bp; Sp1, 821 bp; Sp3, 514 bp;
and GAPDH, 198 bp). After photography of the gels, the intensity of the
corresponding bands was quantified by densitometric scanning using the
ImageQuant software (Molecular Dynamics) and normalized to GAPDH
cDNA levels. For that purpose, the negative of the photograph (665 Polaroid film) was scanned. For quantification of COL2A1,
Sp1, Sp3, and GAPDH cDNAs, we generated an amplification curve as a
function of all PCR cycle numbers that allowed us to determine the
exponential amplification zone for each cDNA. Then, to normalize
COL2A1, Sp1, and Sp3 cDNA levels to GAPDH cDNA
amounts, the number of cycles for each cDNA was selected
approximately in the middle of the linear range of amplification. Thus,
to obtain the histogram located in the bottom part of the reverse
transcriptase-PCR figures, the densitometric values of a particular PCR
cycle number of COL2A1, Sp1, and Sp3 cDNAs were divided
by the densitometric data of GAPDH cDNA obtained for a different
PCR cycle number. If to create the histograms, different PCR cycle
numbers data are used, very slight variations in the extent of TGF-1
effects are observed indicating that the major conclusions drawn are
not infirmed.
Western Blotting--
Western blot analyses of type II collagen,
Sp1 and Sp3, were performed on RAC nuclear extracts as previously
described (40). For quantification, the electrophoregram was scanned
with an image scanner and the relative intensity of detected signals
was measured and analyzed with a computerized image analysis program
(ImageQuant, Molecular Dynamics).
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RESULTS |
TGF-1 Induces an Inhibition of Type II Collagen Production in
Proliferating Chondrocytes--
To examine the effect of TGF-1 on
type II collagen production by RAC, Western blotting analysis was
performed on primary RAC cultures incubated with or without TGF-1 (1 or 3 ng/ml) for 24 h. The study was done on the cell
layer-associated fraction, as this latter contains the major part of
neosynthesized collagen. These experiments were carried out in the
presence or absence of serum in the culture medium during the
incubation period with TGF-1, to minimize any interference with
serum growth factors and to prevent binding of TGF-1 to
2-macroglobulin. Fig. 1
shows that a protein of apparent mass of 250 kDa clearly reacted with the antibody and therefore correspond to type II procollagen. TGF-1
down-regulated type II procollagen production in primary RAC cultures,
whenever the serum was present (47% and 40% for TGF-1 at 1 and
3 ng/ml, respectively) or absent (15% and 60% when TGF-1 was
used at 1 and 3 ng/ml, respectively) in the culture medium, indicating
that a part of TGF-1 remains active and is not partially inactivated
by serum 2-macroglobulin as already demonstrated (61,
62).
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Fig. 1.
TGF-1 inhibits type
II collagen production in proliferating chondrocytes. Primary RAC
(9.6 cm2 dishes) were incubated with or without TGF-1 (1 or 3 ng/ml), in DMEM with or without 10% FCS. After 24 h, protein
extracts were prepared and used in Western blotting experiments as
described under "Experimental Procedures" to detect type II
procollagen using a specific antibody.
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Type II Collagen mRNA Levels of Proliferating Chondrocytes Are
Decreased by TGF-1--
To determine whether TGF-1-decreased
type II collagen synthesis was accompanied by a similar effect at the
transcriptional level, steady-state levels of mRNA were estimated
by semiquantitative reverse transcriptase-PCR on total RNA extracts of
proliferating primary RAC treated or not with TGF-1, in DMEM with or
without 10% FCS. As shown in Fig. 2, the
levels of COL2A1 mRNA were decreased by TGF-1
treatment of RAC cultures with or without 10% FCS.
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Fig. 2.
Type II collagen mRNA levels of
proliferating chondrocytes are decreased under
TGF-1 treatment. 3 µg of total RNA
extracted from proliferative primary RAC, treated with or without 1 or
3 ng/ml TGF-1, in DMEM with or without FCS, were reverse-transcribed
into cDNA using specific antisense primers for human
COL2A1 and GAPDH mRNAs. 15-33 PCR cycles were performed
using conditions as described under "Experimental Procedures." The
products were analyzed by 2% agarose gel electrophoresis in the
presence of ethidium bromide. After photography of the gels under UV
light, densitometric analysis was performed and the amounts of
COL2A1 cDNAs were normalized to GAPDH cDNA levels,
and shown as histograms. To obtain these histograms, the densitometric
values of COL2A1 cDNA obtained after 24 PCR cycles (0%
FCS) or 21 cycles (10% FCS) were divided by the densitometric data of
GAPDH cDNA observed after 27 PCR cycles.
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TGF-1-induced Inhibition of Type II Collagen Expression Is
Specific and Mediated by a Short 63-bp Promoter Fragment Located
Immediately Upstream of the Transcription Start Site--
To further
investigate the molecular mechanisms whereby TGF-1 down-regulates
type II procollagen production and COL2A1 mRNA levels,
the transcriptional activity of the human COL2A1 gene was
assayed using the luciferase reporter gene construct pGL2-basic. To
delineate the sequences implicated in that effect, transient cotransfections were performed in RAC cultures, using several constructs containing deletions in both the promoter and/or first intron regions of the COL2A1 gene. As shown in Fig.
3A, TGF-1 inhibits the transcriptional activity of all of these constructs, from
the largest one (pGL2-3.774 kb) covering 1 kb of the promoter and
~90% of the first intron region, to the shortest, which contains only a proximal 63-bp promoter. These experiments suggest that the
TGF-1-induced inhibition of the COL2A1 gene is mediated
by a 63-bp proximal promoter.
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Fig. 3.
TGF-1-induced
inhibition of collagen type II expression is specific and mediated by a
short 63-bp promoter fragment located upstream the transcription start
site. Primary RAC at 80% confluency were transiently
cotransfected with 10 µg of different COL2A1 reporter
plasmids together with the expression vector pSV40-gal (2 µg)
(panel A, in 0 or 10% FCS-containing DMEM; panel
B, DMEM + 10% FCS; panel C, in DMEM without FCS). These plasmids were
also cotransfected with 15 µg of pCMV5 expression vector with or
without the cDNA encoding a TGF-1 hybrid receptor (panel
B). After 10-15 h of transfection, the medium was changed and the
cells were incubated with or without TGF-1 (3 ng/ml), in DMEM with
or without FCS. 24 h later, the samples were harvested and protein
content, luciferase, and -galactosidase activities were assayed.
Each series of transfections was performed in triplicate.
Transcriptional activity of each construct was expressed as relative
luciferase activity, after correction for both protein amount and
transfection efficiency.
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To check the specificity of TGF-1 effects on human COL2A1
gene transcription, luciferase reporter vectors were cotransfected with
an expression pCMV5 vector with or without the cDNA encoding a
hybrid TGF-1 receptor, TRII/I, functioning in a dominant negative fashion (53). As shown in Fig. 3B, TRII/I
overexpression leads to a substantial increase in transcriptional
activity of all the constructs. The COL2A1 promoter deletion
data demonstrated that the 266-bp promoter is the highest
transcriptionally active promoter in these experimental conditions.
This could be because of the presence, in the last two constructs
(i.e. pGL2-2.367kb and pGL2-1.167kb), of the potential
silencer element already reported (39). Additionally, when the silencer
element was deleted in the pGL2-0.387kb construct, a maximal
transactivating effect of transfected TRI/II was observed. All
together, these results suggest that basal endogenous TGF-1 produced
by RAC already inhibits the transcription of the human COL2A1 gene. As a consequence, when primary RAC were
cotransfected with the TRII/I vector and treated by TGF-1, the
TGF-1-induced inhibition was completely abolished.
To delineate more precisely the TGF-1 inhibitory effect on
COL2A1 gene transcription and render the 63/+47-bp
construct nonresponsive to the cytokine, we generated a 35/+47-bp
plasmid in which the putative responsive GC-box was deleted. As shown in Fig. 3C, the 35/+47-bp reporter construct
is ~11-fold less transcriptionally active compared with the
63/+47-bp plasmid and the TGF-1 inhibitory effect on
COL2A1 gene transcription was lost with this
shortest construct. These data strongly suggest that TGF-1-induced
repression of COL2A1 gene transcription is specific and is
mediated through a proximal 63/35-bp promoter of the
COL2A1 gene.
Delineation of DNA-binding Sites That Mediate the TGF-1
Inhibitory Effect on the 63-bp Fragment of COL2A1 Promoter--
To
determine cis-acting elements that mediate the inhibitory
effect of TGF-1 on type II collagen expression, DNase I footprinting analysis was performed on the 63-bp proximal promoter, using nuclear extracts from primary RAC treated or not by TGF-1. As shown in Fig.
4, several regions of the 63-bp fragment
were found to be protected by proteins present in nuclear extract from
proliferating RAC. They were located between 8 to 63-bp.
30/63-bp sequences also bind Sp1 recombinant protein. The same
cis sequences are also protected by nuclear extracts from
RAC-treated TGF-1, without apparent differences in the intensity and
size of the protected area.
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Fig. 4.
Footprint analysis of the 63-bp
COL2A1 promoter fragment with nuclear extracts from
chondrocyte cultures treated or not by
TGF-1. The
SmaI-HindIII fragment of the pGL2-0.110kb plasmid
was end-labeled to its 5' SmaI extremity, incubated with or
without different nuclear protein extracts, and treated with DNase I. Panel A: lane 1, Maxam-Gilbert G + A sequencing
reaction of the end-labeled probe. Lanes 2 and 3,
DNase I digestion pattern of the naked DNA incubated without nuclear
extracts. Lanes 4-6, DNase I digestion of the probe
incubated with 15, 30, and 45 µg of primary RAC nuclear extracts.
Lanes 7-9, with 15, 30, and 45 µg of nuclear extracts
from RAC treated with TGF-1 (3 ng/ml). Panel B: lane 1,
naked DNA. Lanes 2 and 3, the probe was incubated
with 0.5 pmol of recombinant Sp1. Lane 4, Maxam-Gilbert G + A sequencing reaction of the labeled probe. The protected areas are
indicated by brackets.
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As an attempt to identify the transcription factors that bind this
proximal promoter, EMSA analysis was performed using the wild-type
oligonucleotide sequences identified in DNase I footprint experiments
on the 63-bp promoter fragment and their mutant counterparts (Table
I). Five wild-type double-stranded
labeled oligonucleotides were generated and incubated with nuclear
extract from primary RAC. As shown in Fig.
5A, three major complexes
called a, b+b', and c were formed upon
incubation of three probes (67/+1wt, 50/+1wt, 67/30wt) with
nuclear extracts from RAC, and, in that case, DNA binding activity of
the transcription factors involved in a and b+b'
complexes was decreased when nuclear extracts from RAC treated with
TGF-1 were used. The complex b+b' formed upon incubation
with the 67/+1 and 50/+1 probes has been so-called because two
putative transcription factors are present. b represents the
factor binding between the 50 and 30-bp sequence present in
67/+1, 50/+1, and 67/30 oligonucleotide probes as it will be
concluded subsequently from the data obtained with the 35/+1 wt and
30/15 wt probes. Complex c is nonspecific because it is
not always detected, even when the same RAC nuclear extracts and the
same probes are used (see figure 5, panels D-F), and DNA
competition experiments confirm nonspecific binding (see Fig. 5,
panels B and C).
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Fig. 5.
Gel retardation assays with different
subfragments of the 63-bp TGF-1 responsive
sequence. DNA-protein complexes were analyzed by electrophoretic
mobility shift assay. Panel A, five labeled double-stranded
oligonucleotides that have been identified in footprint experiments
(probes 67/+1wt, 67/30wt, 50/+1wt, 35/+1wt, and 30/15wt)
were incubated with 7.5 µg of nuclear extracts from primary RAC,
treated with or without TGF-1 (3 ng/ml). Note that when the 67/+1
and 50/+1 probes were used, there was an eventuality that two
transcription factors of different natures were implied in the
formation of b+b' complex. With the 67/30 probe, only
one transcription factor was involved and formed complex b,
whereas with the 35/+1 and 30/15 probes, a different factor
called b' belong to this complex (two last panels
in the right part of A). Panels
B-F, DNA competition experiments were carried out with the
indicated molecular excesses (×25, 50, and 100) of wild-type
COL2A1 promoter nonlabeled oligonucleotides or other
consensus DNA binding sequences from known transcription factors (Table
I). Only nuclear extracts from control primary RAC were used.
Panel G, the indicated 32P-labeled probes were
incubated with 7.5 µg of nuclear extracts from chondrocyte cultures
treated with or without TGF-1 (3 ng/ml). C, control
cultures; T, cultures incubated with TGF-1. Panels
B-G, when a probe includes the 44/31 sequence, it binds the
b transcription factor, whereas when a probe contains the
30/15 sequence, b' factor binds to it. When the two
indicated sequences are present in the probe, the complex formed
included b+b' factors. In each panel, protein-DNA complexes
are indicated by an arrow.
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Indeed, with 35/+1wt and 30/15wt probes, only a b'
complex is formed. It was so-called because TGF-1 treatment of the
RAC did not modulate the DNA binding activity of this complex, suggesting that the Sp1-like element localized between 30 and 15 bp
does not have functional transcriptional effects, and that the
b' transcription factor could be potentially different from
b, although both of them migrate in the same position in the
electrophoresis gel (Fig. 5A). These data suggest that if
complexes a and b are specific, the DNA-binding site of trans factors involved in these complexes, which
were shown to display a decrease in binding activity under TGF-1
exposure, is located between 30 to 67-bp sequences. Moreover, it is
highly probable that the transcription factors of complexes
a and b bind to the Sp1-like cis
element located between 41 and 33 bp (5'-GGGGCCGGG-3'), which
centered the 50/30-bp of COL2A1 promoter.
In addition, other EMSA analysis were performed with some wild-type
probes as nonradioactive competitors and mutant probes targeted at
GC-rich sequences (Table I). They demonstrated that a and
b+b' complexes bind specifically to the probes, because the
formation of these complexes was competed away when cold wild-type
probes (50/+1wt, 67/30wt, and 49/-28wt) were present in the
binding reaction, whereas it is not the case with the 35/+1wt and mut
probes. These data confirm that trans factors of
a and b complexes bind probably between 30 and 50 bp (Fig. 5, B and C). The En°1wt
oligonucleotide (+2817/+28451(II)) and its cognate mutant were used,
respectively, as positive and negative control competitors, as we
already demonstrated that this sequence represents a high affinity
Sp1/Sp3 DNA-binding site present in the COL2A1-specific
enhancer (39, 40). Addition of molar excesses of the En°1wt
oligonucleotide prevented formation of the complex a and
b+b', whereas the En°1mut had no effect, indicating that
complex a may involve Sp1 and/or Sp3, whereas complex
b+b' implicates Sp3 and/or another factor binding to the
GC-rich motifs. Complex c formation was not inhibited by
molar excesses of the +2817/+28451(II)wt competitor. In contrast, it
was suppressed when the respective mutant was used, indicating that
binding of the transcription factor(s) involved in this complex is not specific.
To further characterize the transcription factors binding to the 63-bp
promoter, additional EMSA competition experiments were performed using
competitors representing some consensus DNA-binding sites from known
transcription factors. The data showed that the binding activity is
specifically and slightly decreased when competitors containing CG-rich
sequences were used, such as Krox, AP2, and NF-
B wild-type
oligonucleotides, but not by the same molar amounts of the respective
mutant oligonucleotides (Fig. 5, C and
E). Moreover, addition of cold molar excesses
of the EGRwt and mut, and TFIID-wt oligonucleotides did not really
modify the binding to the 50/+1wt or 67/30wt probes of
trans factors included in complexes a or
b or b+b' (Fig. 5D and data not shown).
To further delineate the location of the binding site for a
and b+b' complexes, additional competition experiments were
carried out. As shown in Fig. 5F, the binding of these
complexes to the 50/+1wt probe was abolished when molar excesses of
50/+1wt or 67/30wt cold oligonucleotides were added, whereas the
binding was not competed away when the same molar excesses of cold
35/+1wt and mut, 30/+1wt, and 15/+1wt oligonucleotides were
included in the binding reaction. Similarly, in direct binding gel
retardation experiments, only the 44/19wt, 49/28wt, and
48/32wt probes bound complexes a and b,
whereas they did interact very weakly with the 48/32mut probe (Fig.
5G). In all cases, nuclear extracts from TGF-1-treated
RAC cultures displayed a lower binding activity of complexes
a and b compared with the control nuclear
extracts. When the 30/15wt sequence was used as a probe, a very
slight binding of complex b' was observed and TGF-1 did
not modulate the binding activity. All these results, presented in Fig.
5, suggest that the binding of trans-acting factors
participating in the formation of complexes a and
b is specific, that the transcription factors involved in
these complexes bind with high affinity between 50 and 35-bp in the
COL2A1 promoter, and that TGF-1 decreases binding activity of complexes a and b.
To get further insights into the nature of nuclear proteins binding to
the COL2A1 proximal promoter, antibody interference assays
were performed with specific antibodies. Nuclear extracts from primary
RAC were incubated with 50/+1, 67/30, and 35/+1 wild-type
probes, together with Sp1, Sp3, p50, and p65 subunits of NF-B
antibodies. A supershifted complex formed between Sp1 of nuclear
extracts or human recombinant Sp1 and the probes 50/+1 and 67/30
was detected (Fig. 6, panels
A-C). No supershift occurred with anti-Sp3 antibody incubated
with RAC nuclear extracts, but the DNA probe-Sp3 complexes disappeared,
indicating that the antibody can bind Sp3 (39, 40). Indeed, when
nuclear extracts from untreated RAC were concomitantly incubated with
both Sp1 and Sp3 antibodies, the binding of complex a was
completely abolished (Fig. 6, panels A and C). As
also shown on Fig. 6A, a supershifted complex formed between
the p50 human recombinant subunit of NF-B complex was detected with
all of the probes used (50/+1wt, 67/30wt, and 35/+1wt), but not
with nuclear extracts from RAC. Antibody interference assays were also
carried out using p65 (Fig. 6, B and C), Smad3,
Egr2 (Fig. 6C), and AP2a (not shown) antibodies but no
decrease in a, b, and b+b' complexes binding or even a supershift was observed. Similar data were also obtained with all other antibodies tested on nuclear extracts from
primary proliferative RAC-treated TGF-1 (data not shown). In
conclusion, these experiments suggest that the TGF-1-induced inhibition on COL2A1 expression implies a multimeric complex
involving Sp1 and Sp3 that bind to the 41/33 sequence, but Smad3 is
not involved in this effect, as well as AP2, EGR2, and NF-B.
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Fig. 6.
Characterization of the nature of nuclear
factors mediating the TGF-1 inhibitory effect
on the 63-bp fragment of COL2A1 promoter. Binding
of transcription factors was analyzed by EMSAs. 7.5 µg of nuclear
extracts from untreated primary RAC (RAC P0), as well as recombinant
Sp1 (5 fmol) and p50 (0.7 fmol) have been used in the experiments.
Supershift assays using specific antibodies were performed as indicated
in panels A-C. As in Fig. 5, when the 50/+1 probe was
used, a b+b' complex involving two transcription factors was
formed upon incubation with nuclear extracts. With the 67/30 probe,
only one factor binds to DNA and gives complex b. By
contrast, the 35/+1 probe-nuclear protein complex corresponds to
b' binding to the 30/15 sequence.
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Sp1- and Sp3-binding Sites Mediate the TGF-1-induced
Transcriptional Inhibition of a Nonspecific Promoter in Proliferating
Chondrocytes--
To provide further evidence of the specific role of
Sp1 and Sp3 in the inhibition of COL2A1 gene expression,
transfection experiments were performed with RAC incubated with or
without TGF-1. A reporter vector harboring an oligonucleotide
including four Sp1/Sp3-binding sites, cloned upstream from a thymidine
kinase promoter fused to the luciferase gene, was cotransfected in
primary cultures that were treated with or without the cytokine.
Specificity of the observed transcriptional effects was estimated by
cotransfection of an aspecific reporter plasmid containing four mutated
copies of the Sp1/Sp3 binding element (40). As shown in Fig.
7A, TGF-1 represses the
transcriptional activity of the mcSp1wt reporter vector, whenever the
serum was present or not, whereas the transcription of the respective
mutant plasmid (mcSp1mut) was not modified under the cytokine
treatment.
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Fig. 7.
Sp1- and Sp3-binding sites mediate
TGF-1 transcriptional inhibition of the
COL2A1 gene and a nonspecific promoter in
proliferating chondrocytes. Panel A, primary RAC
cultures treated with or without TGF-1 (3 ng/ml) were transiently
transfected with 10 µg of reporter plasmid (mcSp1wt or mcSp1mut), in
the presence or absence of 10% FCS. The relative luciferase units
represent the mean ± S.D. of three independent samples of a
representative experiment. Panel B, primary RAC cultures
treated with or without TGF-1 (3 ng/ml) were transiently transfected
with 10 µg of the reporter vector pGL2-0.110kb and incubated with or
without 100 nM mithramycin. Panel C, primary
chondrocytes were transfected with 10 µg of reporter constructs
p3TPLux, pGL2-0.387kb, and pGL2-0.110kb. After overnight transfection,
the medium was replaced and the cells were incubated for 24 h in
the presence or absence of TGF-1 (3 ng/ml). At the end of the
incubation period, luciferase activity and protein amounts were
assayed. The relative luciferase units represent the mean ± S.D.
of three independent samples of a representative experiment and were
expressed as % of TGF-1 effect versus respective
control.
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Another approach to confirm these data was to cotransfect the
pGL2-0.110kb vector carrying the 63-bp proximal promoter, which is
implicated in the TGF-1-induced inhibition of COL2A1 gene transcription, in RAC treated with or without TGF-1 in the presence or absence of mithramycin, known as an inhibitor of Sp1/Sp3 binding to
their target DNA-binding site. As shown in Fig. 7B, addition of mithramycin induced an ~100% increase of basal pGL2-0.110kb transcriptional activity, and the TGF-1-induced inhibition was abolished. These data confirmed that the Sp1 and Sp3 DNA-binding motifs
located between 41/33 bp in the COL2A1 proximal promoter mediated the transcriptional TGF-1 down-regulation of that gene in
primary proliferative RAC. Moreover, the TGF-1-induced repression of
gene transcription through a Sp DNA-binding site was also observed when
an aspecific promoter/reporter construct was used.
As further proof of the specificity of TGF-1 effects on
COL2A1 gene transcription in RAC, primary cultures were
transfected with the reporter gene construct p3TPLux, a derivative of
the plasminogen activator inhibitor-1 promoter that is highly
responsive to the cytokine via Smad3. As shown in Fig. 7C,
TGF-1 increases the transcriptional activity of p3TPLux by
~20-fold, whereas it inhibits the transcription of the pGL2-0.387kb
and pGL2-0.110kb constructs containing, respectively, 266 and 63 bp of
the COL2A1 proximal promoter, indicating that the TGF-1
effects on type II collagen gene are specific and independent of Smad3 activation.
Decoy Sp Oligonucleotides Prevent TGF-1 Repression of the
Transcriptional Activity Mediated by the 63-bp COL2A1
Promoter--
Decoy experiments were carried out as an attempt to
demonstrate that the inhibitory effect of TGF-1 on COL2A1
promoter activity is really mediated through a Sp1-binding site. For
that purpose, chondrocytes were transfected with the pGL2-0.110kb
construct together or not with wild-type double-stranded
oligonucleotides bearing a Sp cis-sequence. The
COL2A1 promoter transcription was determined after a 24-h
incubation period in the presence or absence of TGF-1 treatment. A
multicopy (two copies) of the 50/+1wt COL2A1 promoter
sequence that mediated the inhibitory effect of the cytokine through
the 41/33 Sp-binding site was used as a decoy oligonucleotide. When
RAC were transfected with the reporter construct in the absence of
decoy oligonucleotide, TGF-1 caused a 80% decrease in promoter
activity (Fig. 8A). When the
reporter construct was cotransfected with the 50/+1wt
oligonucleotide, the decoy oligonucleotide prevented TGF-1-induced
inhibition of transcription. When the same oligonucleotide was not
transfected, but simply added after the overnight transfection in the
culture medium with or without TGF-1, this oligonucleotide was also
capable of blocking cytokine inhibition of promoter activity. Finally, cotransfection of the decoy sequence with the reporter gene construct, followed by addition of this oligonucleotide in the culture medium gave
the same results as observed in the two preceding experimental conditions. Because the data were similar whenever the decoy sequence was transfected or simply added in the culture medium, we deliberately chose to cotransfect the COL2A1 construction together with
the decoy oligonucleotide. To pinpoint the potential involvement
of the 41/33-binding site that possibly governs the TGF-1
inhibition of COL2A1 gene activity, we used the 67/30
COL2A1 sequence as a competitor oligonucleotide. Results
presented in Fig. 8B indicate that TGF-1 efficiently
decreased the COL2A1 promoter activity by ~60%.
Transfection of the 67/30wt oligonucleotide prevented the
cytokine-induced repression of the promoter activity, whereas the
67/30mut bearing a mutation in the 41/33 sequence, 35/15wt, and mutant (mutation of the two Sp1-binding sites) oligonucleotides were unable to abolish the negative effect of TGF-1 on transcription of the pGL2-0.110kb construct. To further demonstrate that TGF-1 induces its effect through a Sp1-binding sequence, a
107/1351(II)wt multicopy oligonucleotide containing four copies
of a Sp1 consensus binding site previously characterized in the
COL2A1 gene (40) was used as a decoy oligonucleotide.
Similarly, this decoy cis-element prevented the TGF-1
inhibitory effect on COL2A1 promoter activity in a specific
manner, because no modifications of the inhibition of transcriptional
activity under cytokine treatment were observed with the respective
mutant sequence (Fig. 8C). Taken together, the data
presented in Fig. 8 demonstrate that the 41/33 sequence found in
the COL2A1 promoter is responsible for the TGF-1-induced inhibition of transcription of that gene and this element can be used
as a decoy oligonucleotide to prevent this effect.
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Fig. 8.
Decoy oligonucleotides containing
Sp1-DNA-binding sites prevent inhibition of the 63-bp COL2A1
promoter activity induced by TGF-1.
Panel A, primary RAC cultures in 9.6-cm2
dishes were transfected with 10 µg of pGL2-0.110kb construct,
pSV40-Gal (2 µg), and, when indicated, with 30 µg of a multicopy
(two copies) of 50/+1 sequence found in COL2A1 promoter.
TRANSFECTION mentions that the decoy sequence has been
cotransfected with the reporter construct. MEDIUM means that
the oligonucleotide has not been transfected but added to the culture
medium during the incubation period in the presence or absence of
TGF-1. A mixed series of samples were cotransfected with the decoy
oligonucleotide and then after medium replacement, the same sequence
was supplemented in the medium (indication: TRANSFECTION + MEDIUM). After the transfection period, the cells were incubated
for 24 h in DMEM + 10% FCS with (hatched bars) or
without (solid bars) TGF-1 (3 ng/ml). The relative
lucliferase units expressed in percent of TGF-1 effect
versus respective control represent the mean ± S.D. of
three independent samples of a representative experiment. Panel
B, primary RAC in 9.6-cm2 dishes were cotransfected
with 10 µg of pGL2-0.110kb reporter gene construct, pSV40-gal (2 µg), and when indicated with 60 µg of the indicated Sp1 decoy
oligonucleotides. After 10-15 h of transfection, the medium was
changed and the cells were incubated in DMEM + 10% FCS in the presence
(hatched bars) or absence (solid bars) of
TGF-1 (3 ng/ml). 24 h later, luciferase and -galactosidase
activities were determined and corrected for protein amounts. Relative
promoter activity under TGF-1 treatment was expressed in % versus respective control. The relative luciferase units
represent the mean ± S.D. of three independent samples of a
representative experiment. Panel C, primary chondrocytes
were cotransfected and incubated as described in panels A
and B, except that RAC were transfected with 50 µg of
Sp1mcwt and corresponding mutant oligonucleotides. Expression of the
data is identical as in panels A and B.
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TGF-1 Modulates Sp1 and Sp3 Expression in Proliferating
Chondrocytes--
Besides TGF-1 effects on DNA binding activity of
Sp1/Sp3 to the proximal 63-bp COL2A1 promoter, we
searched for a potential effect of the cytokine on the
expression of these two Sp transcription factors. For that purpose,
reverse transcriptase-PCR analysis using total RNA extracted from
primary RAC, treated with or without TGF-1, was performed. As shown
in Fig. 9, the steady-state levels of Sp3
mRNA were increased by TGF-1, but Sp1 mRNA levels were found
to be decreased. To determine whether there was a correlation with the
respective amounts of Sp1/Sp3 proteins, Western blotting experiments
were done. Using nuclear extracts from primary RAC, four major
polypeptides of relative molecular mass 105, 95, and a doublet of
~50-55 kDa were detected with the Sp1 antibody (Fig. 10A, lane 1). The 105-kDa
isoform corresponds to the phosphorylated Sp1 protein, whereas the
95-kDa polypeptide relates to the nonphosphorylated Sp1 (40). The
amount of both forms of Sp1 was decreased by TGF-1 treatment of the
RAC, respectively, by 51 and 27%. The same nuclear extracts were
probed with Sp3 antibody. As shown in Fig. 10A (lanes 4 and 5), a protein of ~105 kDa and a doublet of 60 kDa, similar to that already described (40, 52), were revealed.
TGF-1 had no effect on the amount of Sp3 isoforms, suggesting that
this cytokine increases Sp3 mRNA stability.
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Fig. 9.
TGF-1 decreases Sp1
mRNA steady-state levels, but increases the Sp3 mRNA
steady-state levels of proliferating chondrocytes. 3 µg of total
ARN extracted from primary RAC were reverse-transcribed into cDNA
by using specific antisense primers for Sp1, Sp3, and GAPDH mRNAs.
15-45 PCR cycles were performed under conditions described under
"Experimental Procedures." For normalization of Sp1 and Sp3
cDNA expressions to GAPDH cDNA levels, the densitometric values
obtained after 30 PCR cycles for Sp1 and Sp3 were divided by
densitometric data observed after 25 PCR cycles for GAPDH. If other PCR
cycle numbers are used, very closely related results are obtained
(examples for Sp1 cDNA/GAPDH cDNA ratio: 25 cycles/22
cycles = 2.08; 35 cycles/28 cycles = 1.77; 40 cycles/31
cycles = 1.86). N.T., not treated.
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Fig. 10.
Modulation of Sp1/Sp3 amounts and binding
activity by TGF-1 in proliferating RAC.
Panel A, TGF-1 inhibits Sp1 protein expression,
whereas it does not modulate Sp3 expression in primary RAC. 40 µg of
primary RAC nuclear extracts, treated with or without TGF-1 (3 ng/ml), were separated on a 10% polyacrylamide gel in denaturating
conditions. Recombinant Sp1 protein (0.5 ng) was used as a positive
control (lane 3). Then, proteins were transferred to a
polyvinylidene difluoride membrane, and reacted with polyclonal
antibody against Sp1 (lanes 1-3) and Sp3 (lanes
4 and 5) (1/1000 dilution). Sp1 and Sp3 proteins were
revealed with a peroxidase-coupled secondary antibody (anti-rabbit IgG,
1/1000 dilution) using an ECL Western blot detection kit. The position
of molecular mass standards, expressed in kDa, is indicated.
Panel B, Southwestern analysis of the polypeptides binding
to a Sp1/Sp3 DNA-binding site present in the specific COL2A1
enhancer. Cells were harvested at 80-90% of confluency and nuclear
extracts were prepared as described under "Experimental
Procedures." 40 µg of nuclear extracts from primary RAC treated
with or without TGF-1 (1 ng/ml) were fractionated by SDS-PAGE (10%
gel), transferred to a polyvinylidene difluoride nylon membrane, and
submitted to Southwestern blotting. After a denaturation/renaturation
protocol, membranes were subsequently incubated with 1 pM
+2440/+24851(II) wild type (lanes 3 and 4) and
mutant (lanes 1 and 2) probes as described under
"Experimental Procedures." The position of the molecular mass
standards expressed in kDa is indicated.
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Further evidence of the TGF-1 effects on binding activity of Sp1 and
Sp3 was provided in Fig. 10B, where Southwestern experiments were performed with +2440/+24851(II) wild-type
(lanes 3 and 4) and mutant (lanes 1 and 2) probes representing a cis-element present in the human COL2A1 intronic enhancer. This sequence
includes a Sp1/Sp3-binding element as previously described (39, 40). Three major polypeptides with relative mass of 100, 65, and 55 kDa
bound this sequence. The complex of ~100 kDa corresponded to Sp1 and
Sp3, because we demonstrated that these transcription factors bind this
cis-element in EMSAs performed with or without Sp1 and Sp3
antibodies (40). The slower migrating species most probably reflected
Sp3 binding to the probe because a Sp3 antibody recognized 100-, 60-, and 58-kDa polypeptides (40, 52). TGF-1 induces a 42% decrease in
the binding of the 100-kDa polypeptide to the wild-type probe, whereas
a lower repression of the binding activity of 60 and 58 kDa was caused
by the cytokine. These data suggest that TGF-1 has a much more
marked effect on Sp1 binding activity compared with that of Sp3,
thereby inducing an increase of Sp3/Sp1 ratio.
Effects of Sp1 and Sp3 on the Transcriptional Activity
of the 63-bp Promoter Mediating TGF-1 Inhibition of COL2A1 Gene
Expression--
To further verify the roles played by Sp1 and Sp3 in
TGF-1 down-regulation of human COL2A1 gene expression,
cotransfections were performed in RAC with Sp1 and Sp3 expression
vectors, together with the pGL2-0.110kb luciferase vector mediating the
TGF-1-induced inhibition of transcription. We have previously
demonstrated that Sp1 was a strong activator of the COL2A1
promoter, but no effect was observed under Sp3 overexpression (40). By
contrast, Sp3 overexpression was shown to block the Sp1 induction of
the COL2A1 promoter activity. These effects were mediated by
a short promoter containing a 266-bp region upstream from the
transcription start site of the human COL2A1 gene (40). As
shown in Fig. 11, overexpressed Sp1
strongly activated transcription of the pGL2-0.110kb construct, but Sp3
did not affect significantly the transcription of this plasmid when 10 µg of Sp3 expression vector were cotransfected. Additionally, when
RAC are cotransfected with 20 µg of Sp3 expression vector, the
transcriptional activity of the COL2A1 proximal promoter was
decreased. When the reporter plasmid was cotransfected with a fixed
amount of Sp1 expressing vector and increasing amounts of pRC/CMV/Sp3,
Sp1 induction of COL2A1 transcription was completely repressed by Sp3. Because the transactivating effect of Sp1 being overcome by Sp3 and that Sp3 protein amounts were not modified by
TGF-1 treatment, but its DNA binding activity is slightly decreased
while the cytokine greatly decreased amounts and binding activity of
Sp1, these data further demonstrated that Sp1 and Sp3 proteins play an
important role in mediating TGF-1-induced inhibition of the human
COL2A1 expression in primary RAC.
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Fig. 11.
Sp3 represses Sp1 induction of
COL2A1 gene transcription through the 63-bp promoter
fragment responsive to TGF-1 in primary
RAC. Primary RAC in 9.6-cm2 dishes were cotransfected
with 10 µg of pGL2-0.110kb construct and the indicated amounts of Sp1
and/or Sp3 insertless expression vectors (pEVR2 and pRC/CMV,
respectively) and/or pEVR2/Sp1 and/or pRC/CMV/Sp3. After 10-15 h of
transfection, the medium was changed, and 24 h later the samples
were harvested and protein content and luciferase activity were
assayed. Each series of transfections was performed in triplicate.
Transcriptional activity of each construct was expressed as relative
luciferase activity, after correction for both protein amount and
transfection efficiency. The relative luciferase units (RLU)
represent the mean ± S.D. of three independent samples of a
representative experiment.
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1 (TGF-
,
,
TOP
ABSTRACT
INTRODUCTION
