From the Department of Internal Medicine and Division
of Clinical Research, National Kanazawa Hospital, Kanazawa
920-8650, Japan, the ¶ First Department of Internal Medicine and
§§ Department of Dermatology, Kanazawa
University School of Medicine, Kanazawa 920-8640, Japan, the ** Allergy
Research Center, Juntendo University School of Medicine, Tokyo
113-8421, Japan, the
Division of Cellular
Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam,
The Netherlands, and the ¶¶ Brookdale Center in the
Department of Biochemistry and Molecular Biology, Mount Sinai School of
Medicine, New York, New York 10029
Received for publication, November 20, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Type I collagen is produced predominantly in
mesenchymal cells, but molecular mechanisms responsible for cell
type-specific expression are virtually unknown. During fibrogenic
process in the liver, activated hepatic stellate cells (HSC) are
the main producers of type I collagen, whereas parenchymal hepatocytes produce little, if any, of this protein. We have previously reported that Sp1 and an interacting unknown factor(s) bind to the Type I collagen, the major component of extracellular matrix in
various organs, is produced predominantly in mesenchymal cells such as
fibroblasts, osteoblasts, and myofibroblasts. It is composed of two
We have previously shown that similar regulatory mechanisms control
COL1A2 transcription in skin fibroblasts (8, 9) and
activated HSC (10). The COL1A2 upstream sequence spanning from 313 to
255 sequence of the
2(I) collagen gene (COL1A2)
and play essential roles for basal and TGF-
-stimulated transcription
in skin fibroblasts and HSC. Recently, Smad3 has been shown to bind to
this region, and its interaction with Sp1 has been implicated in
TGF-
-elicited COL1A2 stimulation. The present study
demonstrates predominant binding of Sp3 rather than Sp1 to this
regulatory element in parenchymal hepatocytes. In these cells, this
region did not exhibit strong enhancer activity or mediate the effect of TGF-
. Transfection of HSC with an Sp3 expression plasmid
abolished the COL1A2 response to TGF-
, whereas
overexpression of Sp1 in hepatocytes increased basal COL1A2
transcription and conferred TGF-
responsiveness. Functional and
physical interactions between Sp1 and Smad3, but not between Sp3 and
Smad3, were demonstrated using the bacterial GAL4 system and
immunoprecipitation-Western blot analyses. These results
indicate that cell lineage-specific interactions between GC box binding
factors and Smad protein(s) may account, at least in part, for
differential COL1A2 transcription and TGF-
responsiveness in HSC and parenchymal hepatocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 chains and one
2 chain, which are coordinately expressed but
encoded by the distinct genes,
COL1A11 and
COL1A2, respectively. Transforming growth factor-
1
(TGF-
1, henceforth referred to as TGF-
) plays important roles in
stimulating type I collagen gene expression mainly at the levels of
transcription (1). In the liver, it has been widely accepted that
activated hepatic stellate cells (HSC) showing the morphological and
functional features of myofibroblasts are the main producers of type I
collagen (2, 3). By contrast, parenchymal hepatocytes produce
little, if any, collagen during hepatic fibrogenesis (4, 5). Recent studies by us and others have indicated that both COL1A1 (6) and COL1A2 (7) transgenes are expressed in mesenchymal
cells, but not in parenchymal hepatocytes, after carbon tetrachloride administration into transgenic mice harboring collagen
promoter-reporter gene constructs. These results further confirmed the
minor contribution of hepatocytes to collagen production. However, very
little is known regarding the molecular mechanisms determining
differential type I collagen gene expression in activated HSC and
parenchymal hepatocytes.
313 to
183 is essential for basal transcription of the gene in
the two cell types of mesenchymal origin and mediates stimulatory
effect of TGF-
on COL1A2 transcription. Thus, we designated this region the TGF-
-responsive element (TbRE). Within the TbRE there are at least two distinct sites of DNA-protein interaction, Box 3A (
313 to
286) and Box B (
271 to
255; Fig. 1). Box 3A contains two GC boxes, which
are bound by a ubiquitous trans-activator Sp1 in skin
fibroblasts and HSC (9). An additional GC box is present in the
intervening sequence between Box 3A and Box B. Our previous work has
demonstrated that TGF-
up-regulates COL1A2 transcription
by modifying the interaction between Box 3A-bound Sp1 and an unknown
nuclear factor(s) bound to the neighboring Box B sequence (9, 11).
Recently, it has been shown that Smad3, an intracellular mediator of
TGF-
signal transduction, binds to the CAGACA sequence present in
Box B and stimulates COL1A2 transcription (12, 13).
Furthermore, we have recently demonstrated that a functional
interaction between Sp1 and Smad3/Smad4 is critical for
TGF-
-elicited stimulation of COL1A2 transcription in
fibroblasts (14), whereas others have implicated functional cooperation with p300/CBP in Smad-dependent stimulation of
COL1A2 transcription (15).
View larger version (13K):
[in a new window]
Fig. 1.
Schematic representation of the
COL1A2 promoter sequence and
COL1A2-reporter gene constructs. The
upper part of the figure shows a map of the
COL1A2 promoter sequence from 378 to +58 linked to either
a CAT or luciferase (LUC) reporter gene. Relative positions
of Box 5A (
330 to
297), Box 3A (
313 to
286), and Box B (
271
to
255) as well as the CCAAT and TATA boxes are indicated. Consensus
recognition sequence for Sp1 (GGGCGG) present in Box 3A and between Box
3A and Box B, as well as additional possible Sp1 binding sites (TCCCCC
from
164 to
159 and TCCTCC from
128 to
123) are depicted as
black boxes. A Smad binding element (SBE) present
in Box B is indicated by a hatched box. COL1A2·CAT and
COL1A2·LUC chimeric constructs used in the present study are
schematically presented below.
The present study was designed to determine whether collagen gene
transcription is differentially regulated in activated HSC and
parenchymal hepatocytes and, if so, to study the molecular mechanisms
responsible for this cell type-specific expression. Our results
indicated that, unlike in mesenchymal cells such as skin fibroblasts
and HSC, strong enhancer activity of the 313 to
183 segment and
response to TGF-
were not observed in primary culture of
hepatocytes. Gel mobility shift assays revealed that, whereas Sp1 was
the major Box 3A-bound factor in HSC, Sp3 bound predominantly to this
GC-rich sequence in hepatocytes. Transfection of activated HSC with an
Sp3 expression plasmid abolished the COL1A2 response to
TGF-
. By contrast, overexpression of Sp1 in hepatocytes increased
the basal level of COL1A2 transcription and conferred
TGF-
responsiveness. Functional and physical interactions between
Sp1 and Smad3, but not between Sp3 and Smad3, were demonstrated using
the bacterial GAL4 fusion protein system and
immunoprecipitation-Western blot analyses. Based on these lines of
experimental evidence, we conclude that interaction between Sp1/Sp3
transcription factors and Smad proteins modulates, at least in part,
cell lineage-specific COL1A2 transcription in HSC and
parenchymal hepatocytes.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Adult rat hepatocytes and HSC were prepared by
collagenase perfusion (16) and Pronase-collagenase perfusion (17),
respectively. They were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% (for hepatocytes) or 20% (for HSC) fetal
bovine serum (FBS). More than 95% purity of parenchymal hepatocytes
and more than 90% purity of HSC were confirmed immunohistochemically
using anti-albumin and anti-desmin antibodies, respectively (data not shown). Primary HSC passaged one to three times were used for transfection experiments. CFSC-2G is an activated HSC clone derived from a cirrhotic rat liver induced by carbon tetrachloride injection (18), and the cells were maintained in the same medium supplemented with 10% FBS and non-essential amino acids. Because primary culture of
rat hepatocytes were found to maintain their differentiated phenotype
better on type I collagen-coated dishes (Corning Glass Works, Corning,
NY; data not shown), primary HSC and CFSC-2G cells were plated on the
same substrata for a strict comparison in the experiments shown in Fig.
2. COS-7 cells were obtained from the American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium with 10% FBS.
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Chimeric Constructs-- Plasmids containing different lengths of COL1A2 upstream sequence linked to either a bacterial chloramphenicol acetyltransferase (CAT) gene or a firefly luciferase gene have been previously described (8, 9, 19) and are schematically shown in Fig. 1. A promoterless CAT construct, pBLCAT3 (20), was used as a negative control. An SV40 early promoter region was cloned into pBLCAT3 vector (designated pSVCAT3) and used as a positive control. Expression plasmids used are pCMV-Sp1 (21) kindly provided by Dr. G. Elder, pCMV-Sp3 (22) from Dr. J. Horowitz, and pCMV-Smad3 (23) from Dr. R. Derynck. In these plasmids, either Sp1, Sp3, or Smad3 is expressed under the control of cytomegalovirus (CMV) promoter. An empty CMV-driven expression vector, pcDNA3 (Invitrogen Corp., Carlsbad, CA), was used as a negative control. Bacterial GAL4 fusion protein expression plasmids, pMSp1, pMDNSp1, and pMSp3, which encode the active form Sp1, transactivation domain-deleted Sp1, and active form Sp3, respectively, were generously provided by Dr. Y. Sowa, as well as a control pM plasmid and the pG5-luc reporter construct (24).
Cell Transfection Assays--
Preparation of plasmid DNA for
cell transfection was previously described (8). CFSC-2G cells were
transfected using the calcium phosphate coprecipitation technique (8)
followed by a 15% glycerol shock for 90 s. FuGENE 6 transfection
reagent (Roche Diagnostic Co., Indianapolis, IN) was used for
transfection of primary culture of rat hepatocytes and HSC. In some
experiments, transfected cells were placed in medium containing 0.1%
FBS and treated with 2 ng/ml of TGF- (Collaborative Biomedical
Products, Bedford, MA). Cells were harvested 48 h after
transfection and subjected to either CAT or luciferase assays. Enzyme
activity of the COL1A2·CAT chimeric constructs was normalized
against that of co-transfected pSVXP1, in which the SV40 early promoter
region was cloned into a promoterless luciferase gene construct, pXP1 (25). Transcriptional activity of the COL1A2/luciferase chimeric constructs and pG5-luc reporter construct was normalized against that
of co-transfected pRLCMV (Promega, Madison, WI), in which a Renilla
luciferase gene was driven by a CMV promoter. To avoid competition
between the CMV promoter region of the expression plasmids and that of
pRLCMV vector, the latter DNA was added to the plasmid mixture at
1:1,000 molar ratio against the former. CAT assays and the dual
luciferase assays were carried as previously reported (26) and
according to the manufacturer's protocol (Promega), respectively.
Preparation of Nuclear Extracts and Gel Mobility Shift
Assays--
Nuclear extracts were prepared from culture cells as
previously reported (27). After incubating nuclear extracts with an end-labeled probe, gel mobility shift assays were carried out as
previously described (27). Binding conditions, as well as the sequences
of the Box 5A (330 to
297) and Box 3A (
313 to
286)
oligonucleotides used as either probes or unlabeled competitors, have
been previously described (9). Identical amounts of nuclear proteins
from each cell source were added to the binding reactions. For antibody
interference assays, antibodies against CCAAT/enhancer-binding proteins
(C/EBP
, C/EBP
, and C/EBP
), NF1, or Sp1-related factors (Sp1,
Sp2, Sp3, and Sp4) were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA).
Immunoprecipitation and Western Blot
Analysis--
Immunoblotting of nuclear proteins was performed as
previously described (11) using antibodies against Sp1 and Sp3. To
analyze interactions between Smad3 and Sp1/Sp3, COS-7 cells were
transfected with a Myc-tagged Smad3 expression plasmid (23) together
with an expression vector encoding either Sp1 or Sp3, in the presence or absence of HA-tagged constitutive active TGF- type I receptor (ALK5TD) (23). Forty-eight hours later, whole cell lysates were subjected to immunoprecipitation with anti-Myc antibodies (Santa Cruz
Biotechnology, Inc.), followed by immunoblotting with either anti-Sp1
or anti-Sp3 antibodies. Some cell lysates from the transfected cells
were directly immunoblotted with anti-Myc, anti-Sp1, anti-Sp3, or
anti-HA antibodies (Roche Diagnostic Co.) to confirm expression of
Myc-tagged Smad3, Sp1, Sp3, and HA-tagged ALK5TD, respectively, in the cells.
Statistical Analysis--
Values were expressed as mean ± S.D. Either Student's t test or the Mann-Whitney
U test was used to evaluate the statistical differences
between groups: a p value of less than 0.05 was considered significant.
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RESULTS |
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COL1A2 Transcription and Response to TGF- in HSC and Parenchymal
Hepatocytes--
We first compared COL1A2 transcription and
response to TGF-
between activated HSC (CFSC-2G) and primary culture
of rat hepatocytes. Normalization of the assay data against the
activity of the co-transfected pSVXP1 vector allowed us to make a
comparison between the two cell types, despite the fact that we used
two different methods of transfection. In primary culture of
hepatocytes, transcriptional activity of the
378 and
313COL1A2·CAT constructs containing the TbRE was one-third of that
in CFSC-2G transfectants (Fig. 2A). Deletion of the
378 to
183 sequence did not affect COL1A2 transcription in
hepatocytes: there were no significant differences in the CAT enzyme
activity among the
378,
313, and
183COL1A2·CAT transfectants
(Fig. 2A). This was apparently different from the results
obtained with CFSC-2G cells showing that transcriptional activity of
the
378 and
313COL1A2·CAT constructs was significantly higher
than that of the
183COL1A2·CAT construct (Fig. 2A).
Administration of 2 ng/ml of TGF- into the culture medium resulted
in a significant increase in transcriptional activity of the
378 and
313COL1A2·CAT constructs in CFSC-2G cells, but not in primary
culture of hepatocytes (Fig. 2A). Consistent with these
results, a parallel experiment of Northern blot hybridization did not
show any detectable amount of COL1A2 mRNA in either untreated or
TGF-
-treated hepatocytes (data not shown). Transcription of the
183COL1A2·CAT construct did not show TGF-
responsiveness when
transfected into either CFSC-2G cells or primary culture of hepatocytes
(Fig. 2A).
Similar results were obtained when comparing COL1A2 transcription between early passaged HSC, instead of an immortalized HSC clone CFSC-2G, and primary culture of hepatocytes (Fig. 2B).
Characterization of Nuclear Factors Bound to the COL1A2 Upstream
Sequence in HSC and Parenchymal Hepatocytes--
Because the above
functional assays revealed a major difference in the promoter activity
and TGF- responsiveness of the
378 to
183 segment between HSC
and primary culture of hepatocytes, we next examined the binding of
transcription factors present in the nuclei of these cells to the
COL1A2 upstream sequence. Nuclear extracts prepared from
primary culture of hepatocytes and CFSC-2G cells exhibited similar gel
shift patterns when using the Box 5A oligonucleotide as a probe,
although the intensities and relative ratios of the retarded bands were
somewhat different from each other (Fig.
3A). By contrast, gel mobility
shift assays using the Box 3A probe indicated that nuclear proteins
prepared from primary culture of hepatocytes and CFSC-2G cells
demonstrated slightly different migrating patterns. The faster
migrating complexes obtained with both cell types showed different
mobilities from each other (Fig. 3A).
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Antibody interference assays revealed that anti-C/EBP antibodies
effectively diminished the intensity of the Box 5A-bound complexes,
particularly the fast migrating bands, in both primary culture of
hepatocytes and CFSC-2G cells (Fig. 3B), as previously reported with NIH3T3 fibroblasts (28). None of the anti-C/EBP
, anti-C/EBP
, and anti-NF1 antibodies interfered with the complex obtained with either hepatocytes or CFSC-2G cells (Fig. 3B).
When using the Box 3A oligonucleotide as a probe, anti-Sp1 antibodies completely diminished the slowly migrating complex obtained with CFSC-2G nuclear extracts (Fig. 3B, arrowhead). By
contrast, anti-Sp3 antibodies showed a relatively modest effect on both
slowly and faster migrating complexes. On the other hand, in the case
of primary culture of hepatocytes, anti-Sp3 antibodies completely diminished the intensity of the fast migrating Box 3A-bound complex (Fig. 3B, arrowhead), whereas anti-Sp1 antibodies
had a modest influence on both slowly and faster migrating complexes.
Neither anti-Sp2 nor anti-Sp4 antibodies interfered with the complex
formation with Box 3A probe (Fig. 3B).
To analyze semi-quantitatively the amounts of Sp1 and Sp3 proteins present in HSC and parenchymal hepatocytes, nuclear proteins prepared from both cell types were subjected to Western blot analyses using anti-Sp1 or anti-Sp3 antibodies. The results indicated that a larger amount of Sp1 protein was detected in nuclear extracts prepared from freshly isolated HSC than in those from hepatocytes (Fig. 3C). Conversely, more Sp3 protein was present in nuclei from parenchymal hepatocytes than those from HSC (Fig. 3C). Likewise, nuclear extracts prepared from CFSC-2G cells contained a larger amount of Sp1 and less Sp3 protein as compared with hepatocyte nuclei (data not shown).
Effects of Overexpression of Sp1 or Sp3 on COL1A2 Transcription in
Activated HSC and Parenchymal Hepatocytes--
The gel mobility shift
assays and Western blot analyses of nuclear proteins indicated a
difference in the relative amounts of Box 3A-bound Sp1 and Sp3 between
CFSC-2G cells and primary culture of hepatocytes. Thus, we next
examined the effects of overexpression of Sp1 or Sp3 on
COL1A2 transcription in both cell types. Transfection of
CFSC-2G cells with an Sp1 expression plasmid significantly increased
basal transcription of the 378COL1A2·LUC construct (Fig.
4). TGF-
treatment further increased
COL1A2 transcription in Sp1-transfected cells.
Interestingly, overexpression of Sp1 in primary culture of hepatocytes
not only increased basal COL1A2 transcription but also
conferred TGF-
responsiveness to the cells (Fig. 4).
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In contrast to these stimulatory effects of Sp1 overexpression,
transfection of CFSC-2G cells with an Sp3 expression plasmid did not
affect the basal transcription levels (Fig. 4). More importantly, overexpression of Sp3 in CFSC-2G cells completely abolished
TGF--elicited COL1A2 stimulation (Fig. 4).
Functional Interaction between Sp1 and Smad3 in Stimulating COL1A2
Transcription--
We next examined the effects of co-transfecting
either Sp1 or Sp3 expression vector together with an expression plasmid
encoding Smad3, an intracellular mediator of TGF- signal
transduction. Overexpression of Smad3 in CFSC-2G cells significantly
increased transcription of the
378COL1A2·LUC construct (Fig.
5A). Whereas co-transfection
with an Sp1 expression plasmid resulted in a further increase in
378COL1A2·LUC transcription, overexpression of Sp3 did not affect
Smad3-stimulated COL1A2 transcription (Fig.
5A).
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Because there are at least two more Sp1 binding sites downstream of the
TbRE (Fig. 1; Refs. 29, 30), we also performed co-transfection
experiments using the 378COL1A2·LUC construct containing an
internal deletion of the
183 to
108 segment (
378COL1A2del·LUC in Fig. 1). Basal transcriptional activity of this internally deleted
construct was approximately one-fourth of that of the parental
378COL1A2·LUC construct (Fig. 5A), indicating that the two Sp1 binding sites present in the
183 to
108 segment are also
important for basal COL1A2 transcription. However,
co-transfection of Smad3 and Sp1 expression plasmids resulted in the
same level (~3-fold) of transcriptional activation with both
378COL1A2·LUC and
378COL1A2del·LUC constructs (Fig.
5A). It is therefore suggested that Smad3 might interact
with the TbRE-bound Sp1, rather than with the downstream Sp1, to
mediate TGF-
-elicited COL1A2 stimulation.
Functional interaction between Sp1 and Smad3, but not between Sp3 and Smad3, was further confirmed using the bacterial GAL4 system. Transfection of CFSC-2G cells with an expression plasmid encoding GAL4 DNA binding domain-fused Sp1 protein (pMSp1) increased transcription of the pG5-luc reporter gene ~18-fold. Co-transfection with a Smad3 expression plasmid further increased Sp1-stimulated transcription, although Smad3 by itself had no effect on transcription (Fig. 5B). By contrast, transfection with an expression plasmid encoding a transactivation domain-deleted Sp1 (pMDNSp1) resulted in less than 4-fold increase in pG5-luc transcription, and it was not further stimulated by co-transfection with a Smad3 expression plasmid (Fig. 5B). On the other hand, overexpression of an Sp3·GAL4 fusion protein (pMSp3) stimulated transcription 8-fold, which was not affected by co-transfection with a Smad3 expression plasmid (Fig. 5B).
Physical Interaction between Sp1 and Smad3--
In the last set of
experiments, we examined physical interactions between Sp1 and Smad3
and between Sp3 and Smad3 using immunoprecipitation followed by Western
blot analysis. We first attempted to examine interactions between
endogenous GC box binding factors and Smad3 using CFSC-2G cells, but
failed to detect any co-immunoprecipitated proteins (data not shown).
It was not clear whether this was because of a lack of interaction
between Sp1/Sp3 and Smad3 or because of relatively small amounts of
these proteins present in CFSC-2G cells. We therefore transfected COS-7
cells with a Myc-tagged Smad3 expression plasmid together with either
Sp1 or Sp3 expression vector, in the presence or absence of HA-tagged
constitutive active TGF- type I receptor (ALK5TD). Direct
immunoblotting of whole cell lysates with anti-Myc, anti-Sp1, or
anti-Sp3 antibodies confirmed expression of Myc-tagged Smad3, Sp1, and
Sp3, respectively, in transfected COS-7 cells (Fig.
6). In the absence of ALK5TD,
anti-Sp1 antibodies hardly detected immunocomplexes, which had been
first precipitated with anti-Myc antibodies recognizing
Myc-tagged Smad3. By contrast, overexpression of ALK5TD markedly
enhanced the physical interaction between Sp1 and Smad3 (Fig. 6),
indicating that the interaction is TGF-
-dependent. On
the other hand, immunoblotting using anti-Sp3 antibodies failed to
detect Smad3-bound Sp3, either in the presence or absence of ALK5TD
(Fig. 6).
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DISCUSSION |
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In this study, we demonstrated that different molecular mechanisms
control COL1A2 transcription in activated HSC and
parenchymal hepatocytes. Our experimental data indicated the
differential roles of Sp1 and Sp3 in COL1A2 regulation. They
also suggested that, in parenchymal hepatocytes, predominant binding of
Sp3 to the Box 3A sequence and a lack of interaction with Smad3 may
account, at least in part, for relatively low levels of
COL1A2 transcription and loss of TGF- responsiveness. It
should be noted that, although we confirmed more than 95% purity of
hepatocytes, there still remained a small number of HSC contaminated in
the culture (31), which may respond to TGF-
. Despite possible
underestimate of the results because of this contamination of HSC, cell
transfection assays clearly indicated that the COL1A2
promoter containing the TbRE did not show TGF-
responsiveness when
transfected into primary culture of hepatocytes.
Sp1 and Sp3 are closely related proteins with very similar structural features (32). They bind to the common GC-rich sequence named the GC box with the same specificity and affinity and regulate gene transcription (33). However, Sp3 often acts as a transcriptional repressor by competitively binding to the Sp1-bound GC box sequences (33) and/or by expressing internally initiated Sp3 proteins functioning as potent inhibitors of Sp1/Sp3-mediated transcription (34). It is now recognized that Sp3 can either activate or repress transcription of target genes depending on the cell type, the context of DNA binding sites, and the interactions with other nuclear factors (35).
It has been previously reported that both Sp1 and Sp3 stimulate
COL1A2 transcription when transfected into
Drosophila Schneider cells lacking endogenous Sp
transcription factors (36). By contrast, it has been shown by others
that co-transfection of an Sp3 expression plasmid inhibits
Sp1-stimulated COL1A1 transcription in the same insect cells
(37). However, neither of the studies examined the effects of Sp1 or
Sp3 on TGF--elicited COL1A2 stimulation. The present
study clearly demonstrated that, although Sp3 functioned as a weak
trans-activator of transcription in the bacterial GAL4 system, it did not increase basal levels of COL1A2
transcription when transfected into CFSC-2G cells. More importantly,
overexpression of Sp3 abolished TGF-
responsiveness in CFSC-2G
cells. Different behaviors of Sp3 in regulating COL1A2
transcription in Drosophila Schneider cells and mammalian
CFSC-2G cells might be attributed to the presence of interacting
factor(s) in the latter cells (35). On the other hand, overexpression
of Sp1 in primary culture of hepatocytes conferred TGF-
responsiveness with regard to COL1A2 transcription.
A family of proteins termed Smad have been identified (38) and found to
play important roles in the intracellular signal transduction pathways
of the TGF- superfamily members (22). Some of them, Smad3 and Smad4,
have been shown to bind to the so-called CAGACA sequence present in the
promoters of several TGF-
-inducible genes including plasminogen
activator inhibitor-1 (39), junB (40),
p21WAF1/Cip1 (41), and Smad7 (42).
Box B of the COL1A2 promoter also contains a CAGACA sequence
(
265 to
260) (39, 40), and it has been shown that Smad3 binds to
this sequence in vitro and stimulates COL1A2
transcription (12, 13).
We have recently revealed that an interaction between Sp1 and Smad3 is
critical in mediating the stimulatory effect of TGF- on
COL1A2 transcription in NIH 3T3 fibroblasts (14). We
demonstrated a functional interaction between Sp1 and Smad3/Smad4 using
co-transfection experiments. In addition, it was found that, despite
the presence of CAGACA sequence in Box B, recombinant Smad3 and Smad4
proteins did not bind to the
313 to
255 sequence by themselves. Nor
could they stimulate COL1A2 transcription in fibroblasts
stably transfected with an expression plasmid encoding the
dominant-negative form of Sp1 (14). Only in the presence of Sp1
protein, Smad3 and Smad4 were able to bind to this sequence and
synergistically stimulate COL1A2 transcription. These data
suggest the possibility of physical interactions between Sp1 and
Smad3/Smad4 (14). The present study further confirmed these functional
and physical interactions between Sp1 and Smad protein(s) in activated
HSC by utilizing the bacterial GAL4 system and co-immunoprecipitation
experiments. It also revealed that similar interactions were not
observed between Sp3 and Smad3.
A recent study has revealed that the glutamine-rich transactivation
domain of Sp1 and the MH1 domain of Smad3 mediate the interaction
between these two proteins bound to the
p21WAF1/Cip1 gene promoter, which is stimulated
by TGF- (43). Consistent with these results, the present study
showed that co-transfection of an expression plasmid encoding
transactivation domain-deleted Sp1 together with a Smad3 expression
vector did not increase transcription in the bacterial GAL4 system. It
has been previously reported that the glutamine-rich transactivation
domain of Sp1 cannot be replaced by the homologous sequence of Sp3
(33). Taken together, it is conceivable that a lack of interaction
between Sp3 and Smad3 may account, at least in part, for the loss of
TGF-
responsiveness of COL1A2 transcription in
parenchymal hepatocytes.
It has been shown that Smad2, another TGF- responsive Smad, is also
capable of interacting with Sp1 (43, 44). Our recent study revealed
that Smad2 did not bind to the TbRE of the COL1A2 promoter
(14). Nor did transfection of NIH 3T3 cells with a Smad2 expression
plasmid stimulate basal COL1A2 transcription (14). On the
other hand, under a certain experimental condition, overexpression of
Smad2 in primary culture of skin fibroblasts markedly increased
TGF-
responsiveness of COL1A2 transcription without
affecting basal transcription level (45). Functional roles of
Smad2 in TGF-
-stimulated COL1A2 transcription have not been fully understood, and experiments are in progress to clarify possible contribution of Smad2 to cell lineage-specific
COL1A2 transcription.
In conclusion, the present study is the first to demonstrate, at the
molecular level, differences in cell lineage-specific regulation of
COL1A2 transcription in activated HSC and parenchymal hepatocytes. It illustrates that two members of the GC box binding transcription factor family participate in regulation of
COL1A2 transcription through differential interaction with
Smad3. These results lead to not only better understanding of
regulatory mechanisms responsible for cell type-specific gene
expression but also the development of novel therapeutic means for
fibrotic diseases in various organs by suppressing pathologically
activated collagen gene transcription.
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ACKNOWLEDGEMENTS |
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We thank Drs. Rik Derynck, Gregory A. Elder, and Jonathan M. Horowitz for providing us with valuable expression plasmids and to Dr. Yoshikazu Sowa for his generous gift of GAL4 binding domain fusion constructs as well as useful discussion. We also thank Drs. Scott L. Friedman, Francesco Ramirez, Marcos Rojkind, and Shizuko Tanaka for their helpful advice and critical suggestions.
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FOOTNOTES |
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* This work was supported in part by a research grant from the Scleroderma Research Committee of the Ministry of Health and Welfare, Japan (to Y. I.), by a grant-in-aid for Cancer Research from the Ministry of Health and Welfare, Japan (to Y. I.), by a grant from The Netherlands Organization for Scientific Research (to P. t. D.), and by Grant R01 AA12196 from the National Institute on Alcohol Abuse and Alcoholism (to P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Internal Medicine and Division of Clinical Research, National Kanazawa Hospital, 1-1 Shimoishibiki-machi, Kanazawa 920-8650, Japan. Tel.: 81-76-262-4161; Fax: 81-76-263-3450; E-mail: inagaki@kinbyou.hosp.go.jp.
Present address: The Fourth Division, Osaka Bioscience Inst.,
Suita, Osaka 565-0874, Japan.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010485200
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ABBREVIATIONS |
---|
The abbreviations used are:
COL1A1
and COL1A2, genes coding for the 1 and
2 chains of
type I collagen, respectively;
CAT, chloramphenicol acetyltransferase;
C/EBP, CCAAT/enhancer-binding protein(s);
CMV, cytomegalovirus;
FBS, fetal bovine serum;
HSC, hepatic stellate cells;
TbRE, TGF-
-responsive element;
TGF-
, transforming growth factor-
;
HA, hemagglutinin.
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