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INTRODUCTION |
Hepatocyte growth factor
(HGF)1 is expressed in the
stromal cell compartment of a variety of tissues and acts on
neighboring epithelial cells in a paracrine fashion. HGF stimulates
cell growth, cell motility, and morphogenesis on its target cells
through binding and activating its specific tyrosine kinase cell
surface receptor known as Met (1-5). Gene knockout studies have shown
that HGF has an essential role in liver growth during embryonic
development (6, 7). In adulthood, HGF also plays an important role in supporting the maintenance and renewal of cells in various organs such
as liver, lung, and kidney.
HGF gene expression is regulated by extracellular cues such as
hormones and cytokines (8-12). In vivo, the level of HGF
mRNA dramatically increases in response to cell loss to facilitate tissue regeneration. For example, HGF mRNA increases in the liver as well as in distal organs such as lung and spleen after loss of
hepatic tissue induced by partial hepatectomy or hepatotoxin treatment.
Similarly, after injury to the lung or kidney (by toxins or by surgical
procedures such as unilateral pneumonectomy or nephrectomy,
respectively), HGF gene expression is induced in these organs to
promote their regeneration (13, 14).
Several studies have shown that HGF gene expression is controlled
tightly at the transcriptional level. In vivo analyses of HGF gene promoter regulation in transgenic mice (15) as well as in
in vitro transient transfection studies have shown that important regulatory element(s) exist in the proximal promoter region
of the HGF gene (16-20). In recent studies, we have partially characterized this region and revealed that it harbors a composite element located at position
260 to
230 bp from the transcriptional start site. We showed that this composite site binds to members of the
nuclear factor 1 (NF1), activating protein-2 (AP2), and upstream
stimulatory factor (USF) families to regulate HGF gene transcription.
Functional studies show that NF1 and AP2 suppress the activity of the
HGF gene promoter whereas USF has an activating function (21, 22).
In the present studies, we discovered that this region (
260 to
230)
also harbors a functional PPAR
-responsive element at
246 to
233
bp from the +1 site of the HGF gene promoter having the sequence
GGGCCAGGTGACCT. This is apparently a novel PPAR
site, which has an
inverted RGGTCA motif with two spacer (IR2), rather than the
well described DR1 (direct RGGTCA motif with one spacer) (23, 24). Gel
mobility shift and supershift assays using nuclear extracts from
various sources such as whole liver, isolated non-parenchymal
liver cells, fibroblast cell lines, and in vitro translated
PPAR
1 and PPAR
2 protein demonstrated that this element binds to
the nuclear receptor PPAR
family. Additionally, we found that
COUP-TF, an orphan nuclear receptor, also binds to this nuclear hormone
binding site and negates the stimulatory effects of PPAR
. Functional
studies revealed that PPAR
, with its ligand, 15-deoxy-PGJ2,
strongly stimulates HGF promoter activity. On the other hand, NF1 and
AP2 transcription factors repress the stimulatory function of PPAR
by competing with PPAR
for their individual overlapping binding
sites present within this composite element. Moreover, for the first
time, our studies demonstrated that the PPAR
ligand, 15-deoxy-PGJ2,
induces the expression of the endogenous HGF mRNA and protein in
cultured fibroblasts.
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MATERIALS AND METHODS |
Plasmids--
0.7 mouse HGF-CAT promoter construct (
699 to +29
bp) was described in an earlier work from our laboratory (16). 0.7 HGF-CAT/NF1-M, in which the NF1 binding site was mutated, was created
by a sequential PCR mutagenesis method (21). COUP-TFI and AP2
expression vectors were gifts from Dr. M.-J. Tsai, Baylor Medical
College, and Dr. R. Buettner at the University of Regensberg, Germany,
respectively. COUP-TFI in vitro expression vectors were from
Dr. Xiao-Kan Zhang, University of San Diego, San Diego, CA. Dr.
Bin Gao at the Medical College of Virginia generously provided the
NF1/X expression vector. Mouse PPAR
1, PPAR
2, and PPAR
expression vectors, as well as RXR
, were generous gifts from Dr.
Bruce Spiegelman, Dana Farber Cancer Institute, Harvard Medical School,
Boston, MA.
Preparation of Nuclear Extracts--
Mouse NIH3T3 and 3T3-L1
fibroblasts were originally obtained from the American Type Culture
Collection (Manassas, VA) and cultured in the conditions
suggested by the American Type Culture Collection. Cells were grown to
~90% confluence, washed twice with cold phosphate-buffered saline,
and scraped with a rubber policeman in the same buffer. Nuclear protein
extracts were prepared as described previously (20). Rat liver
non-parenchymal cells were prepared from collagenase-perfused rat liver
followed by centrifugation at 50 × g to remove
hepatocytes and were provided by Dr. G. K. Michalopoulos in our
department. They were used to prepare nuclear protein extract.
A rat liver stellate cell line (Ito cell line) was kindly provided by
Dr. M. Rojkind from the Albert Einstein College of Medicine and
cultured as described previously (25, 26). For preparation of mouse
liver nuclear protein extracts, livers were removed from mice and
homogenized in buffer A containing protease inhibitors (20). The nuclei
were collected, and the nuclear proteins were extracted by the same
method as described above.
DNA Transfection and CAT Assay--
Mouse 3T3-L1 fibroblasts
were cultured in 6-well plates for 24 h and then transfected with
various mouse HGF promoter-CAT chimerical plasmids using the DNA
calcium phosphate method according to the instructions of the CellPhect
transfection kit (Amersham Pharmacia Biotech) as described
previously (19). The
-galactosidase reference plasmid pCH110
(Amersham Pharmacia Biotech) was used as an internal control for
monitoring the transfection efficiency and normalizing the data
accordingly. The amount of plasmid used per well in transfection was as
follows: 5 µg of one chimerical CAT construct and 1 µg of pCH110.
After coprecipitation of the cells with DNA-calcium phosphate for
16 h in serum-containing medium, the cells were washed twice with
serum-free medium and kept in serum-containing medium for an additional
24 h before harvesting for determination of CAT activity. CAT
activity was determined as described previously (19, 20). Transfections were performed at least three separate times with two independent preparations of purified plasmid DNA.
For cotransfection with COUP-TFI, AP2, NF1/X, RXR
, and PPAR
1 in
3T3-L1 cells, the amount of plasmid used per well in transfection was
as follows: 5 µg of one chimerical CAT construct, 1 µg of pCH110,
and different amounts of expression plasmids. The total amount of DNA
per well was equilibrated by addition of corresponding empty (no
insert) expression plasmid.
Oligonucleotides and Antibodies--
The following
oligonucleotides and antibodies were purchased from Santa Cruz
Biotechnology, Inc. and used in gel mobility shift and
supershift assays: NF1, 5'-TTTTGGATTGAAGCCAATATGATAA-3'; USF,
5'-CACCCGGTCACGTGGCCTACACC-3'; RXRE (DR-1),
5'-AGCTTCAGGTCAGAGGTCAGAGAGCT-3'; and anti-PPAR
, anti-PPAR
,
anti-Sp1, anti-RXR
, anti-thyroid hormone, anti-COUP-TF (which
can react with both COUP-TFI and COUP-TFII), anti-nerve growth
factor-induced B, and anti-vitamin D receptor antibodies.
Gel Retardation Assays--
The double-stranded oligonucleotides
used in gel mobility shift assays were labeled with
[
-32P]dCTP by end labeling with the Klenow
fragment of DNA polymerase. The labeled oligonucleotide probes were
then gel-purified and used in gel mobility shift assays as described
previously (21). Two µg of poly(dI-dC) (Amersham Pharmacia Biotech)
were used as the nonspecific competitor in 10 µl of reaction mixture.
When antibodies for supershift were used, they (1 µl) were incubated with nuclear extracts at room temperature for 20 min before performing the DNA binding shift assays. The binding reactions were carried out at
room temperature for another 20 min before loading onto 5%
nondenaturing polyacrylamide (19:1, acrylamide/bisacrylamide) gels. The
amount of the nuclear protein extract used in each reaction was about 4 µg, and that of the labeled probe was between 0.2 and 0.4 ng. For
competition experiments, a 100-fold molar excess of unlabeled
oligonucleotides was included in reaction mixtures. Gels were run in
0.5× TBE buffer (0.045 M Tris borate, 0.001 M EDTA) at a constant voltage of 210 V, dried, and autoradiographed with
intensifying screens.
RNA Isolation and Analysis--
Total RNA was isolated by using
RNAzol B solution (Cinna/Biotecx, Friendswood, TX) according to the
manufacturer's instructions. The RNA concentration was determined by
measuring the absorbance at 260 nm. For quantitative RT-PCR, 1 µg of total RNA was reverse-transcribed by using AMV reverse
transcriptase (Roche Molecular Biochemicals) in reverse
transcriptase reaction and amplified for 25 cycles (the number
of cycles were optimized to ensure that quantitative assessment could
be performed) with Taq DNA polymerase (Roche Molecular
Biochemicals) per the manufacturer's instructions using primers specific to HGF (sense, 5'-ATCAGACACCACACCGGCACAAAT-3'; antisense, 5'-GAAATAGGGCAATAATCCCAAGGAA-3') or
-actin
(CLONTECH Laboratories).
-actin served as an RNA
integrity and normalization control.
In Vitro Transcription/Translation--
A transcription
and translation-coupled reticulocyte lysate system was used to prepare
in vitro translated COUP-TFI, mouse PPAR
1, PPAR
2,
PPAR
, and mRXR
proteins from their cognate expression vectors
driven by the T7 promoter (27). Transcription/translation reactions
were carried out in a 50-µl reaction volume as recommended by the
supplier (Promega). The authenticity of translation products was always
confirmed in parallel experiments by including radioactive [35S]methionine and radioactive
[35S]cysteine in the translation reaction followed by
SDS-polyacrylamide gel electrophoresis and autoradiography. Translation
reactions using empty vectors were always included as negative controls in these analyses, including the gel shift assays, to exclude potential
false signals. Translation products were stored at
80 °C. For each
gel mobility shift assay, about 1 µl was used from each translated reaction.
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RESULTS |
Identification of a Binding Site for PPAR
Transcription Factor
in the HGF Gene Promoter--
Our recent studies of the HGF promoter
revealed that the proximal promoter region at position
260 to
230
is a multifunctional composite site to which different transcription
factors such as NF1, USF, and AP2 can bind and regulate HGF gene
transcription (21, 22). During those studies, we found that distinct
factor(s) also specifically bind to this element that are not related
to the NF1, USF, or AP2 families (21). Within this 30-bp region (
260
to
230) of the promoter, we identified RGGTCA hexanucleotide motifs,
which are potential binding sites for the nuclear hormone receptor
superfamily (23, 28). To further characterize the composite binding
site and its cognate transcription factors, we radiolabeled the HGF
promoter composite element (
260 to
230) with 32P and
used this oligonucleotide as a probe to perform gel mobility band shift
assays. In these experiments, nuclear protein extracts from 3T3-L1 and
NIH3T3 fibroblast cell lines were used. As shown in Fig.
1, this DNA element forms several
specific complexes, of which the major complex(es) (shown by a
large arrow head) are efficiently abrogated by an excess
amount of binding site for NF1 (compare lane 2 with
lane 4 and lane 7 with lane 9,
respectively). The remaining two complexes, which we have labeled in
the figure as C1 and C2, formed by nuclear protein extract from NIH3T3
cells, are abolished by an excess amount of USF and nuclear hormone
binding sites (RXRE), respectively (Fig. 1, lanes 9-11). We
have previously shown by super shift assays that the major complex(es)
C contain NF1 isoform(s) and that the C1 complex is composed of USF1
and USF2 isoforms (21). As shown in Fig. 1, 3T3-L1 fibroblasts do not
have detectable C1 and only form the C2 complex, which is totally
abrogated by RXRE (DR1, direct AGGTCA repeat with one spacing) (Fig. 1,
lane 6). Thus, these results extend our previous observation
and demonstrate that the complex C2 may contain nuclear hormone
receptor(s), because it can be competed by a binding site having an
AGGTCA motif (please see Ref. 21). To better define the binding site
for complex C2, several mutant versions of the promoter element were
synthesized (see Fig. 2E for
nucleotide sequences) and used as competitors in electrophoretic
mobility band shift assays using NF1-depleted 3T3-L1 nuclear protein
extracts. As depicted in Fig. 2A, deletion of up to 11 base
pairs from the 5' region did not have a notable effect on the binding
activity (because these oligonucleotides still effectively competed
with the radiolabeled wild-type probe; Fig. 2A, lanes
3 and 4, oligonucleotides designated S1 and S2).
However, when six nucleotides from the 3' region were truncated, the
resulting oligonucleotide totally lost its competitive ability (Fig.
2A, lane 5). These results revealed that the
binding site for C2 should be between
249 and
230. Close
examination of the promoter element (
249 to
230) indicated that it
harbors a potential RGGTCA inverted repetitive sequence separated by
two nucleotides (IR2) (Fig. 2B, shown by arrows).
The AGGTCA site is known as the perfect nuclear hormone binding
half-site, although in almost every case, it is a variation of this
sequence known as imperfect motif (28). Indeed, comparison of the
nucleotide sequence of this 31-base pair element among the mouse, rat,
and human HGF promoters revealed perfect conservation of this site
(Fig. 2B). To define the binding site for the C2 complex in
more detail, several mutated oligonucleotides were synthesized and used
in gel shift competition assays, as above, using fibroblast nuclear
extracts (Fig. 2, C and D). Mutations in the
AGGTCA half-site in the 3' region abolished the binding capability
(mutant oligonucleotides named M4 and M5 in which AGGTCA is mutated to
AGGGAC and CTTTCA, respectively) (Fig. 2C, lanes 6 and 7). Conservative mutations in the 5'-half-site
RGGTCA motif (GGGCCA to AAGCCA or GGGAAA and also mutations in the two
spacers (GG to AT) (oligonucleotides named M1, M2, and M3,
respectively)) did not dramatically affect binding, because
these oligonucleotides still competed with the labeled probe for
binding (Fig. 2C, lanes 3-5). However, mutation
in the 5'-half-site that totally changes the hexanucleotide from GGGCCA
to TTTGGC completely abolished its binding activity (Fig.
2D, lane 8). To define the role of the two
spacers in the IR2 configuration, we generated additional mutant
oligonucleotides as indicated in Fig. 2E and used them as
competitors. As shown in Fig. 2D, deleting of one or both
spacer nucleotides (GG) dramatically reduced the binding activity (Fig. 2D, lanes 5 and 6). On the other hand,
mutating the two putative spacers from GG to TT did not affect the
binding activity (Fig. 2D, lane 7). These results
implied that the RGGTCA half-sites and the two-spacer configuration are
important for binding of complex C2 to this region. As mentioned above,
it is well known that the RGGTCA is the binding half-site for some
members of the nuclear receptor superfamily (23, 28). Therefore,
several antibodies against the members of the nuclear receptor
superfamily were used to identify complex C2 by supershift assays using
fibroblast nuclear extract; these antibodies were against retinoic acid
receptor, OR-1, thyroid hormone receptor, COUP-TF, vitamin D
receptor, and nerve growth factor-induced B. None of these antibodies
reacted with the C2 complex (data not shown). Further analysis using
additional antibodies against other nuclear receptors revealed that
anti-PPAR
antibody reacted with and totally supershifted complex C2
formed by the fibroblast nuclear extracts (Fig.
3, lanes 2 and 6).
Neither anti-RXR
antibody nor an unrelated antibody (anti-Sp1; see
Fig. 3, lanes 4 and 8) reacted with complex C2.
Taken together, the data clearly show that PPAR
binds to the
HGF promoter element located at
246 to
233 bp from the
transcription start site.

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Fig. 1.
Identification of a nuclear receptor binding
site in the composite regulatory element in the HGF promoter
region. Synthetic oligonucleotides corresponding to the
31-bp DNA fragment from 260 to 230 bp in the HGF gene promoter were
labeled with 32P and incubated with nuclear protein extract
(NPE) from NIH3T3 and 3T3-L1 fibroblasts. The binding
reactions were performed at room temperature for 20 min in the presence
of the nonspecific competitor poly(dI-dC). Consensus NF1, USF, and RXRE
(DR1), as well as unlabeled self-double-stranded oligonucleotides, were
used as competitors. The complexes labeled C and indicated
by a large arrow contain NF1 isoforms; the complex labeled
C1 contains USF1 and USF2 (21); and the C2 complex contains
a putative nuclear receptor; F denotes free
32P-labeled oligonucleotide probe.
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Fig. 2.
Characterization of the binding motif(s) for
the nuclear receptor in the HGF promoter regulatory region.
A, truncated (shortened from 5' or 3' ends as indicated in
E) oligonucleotides S1, S2, and S3 corresponding to the
promoter element were synthesized and used as competitors in gel
mobility competition assays using NF1-depleted nuclear protein extract
from 3T3-L1 fibroblasts and the wild-type HGF promoter element ( 260
to 230) as a probe. B, analysis of the potential RGGTCA
motifs within the regulatory region (from 260 to 230 bp) of the
mouse HGF gene promoter. RGGTCA inverted repeats separated by two
nucleotides (IR2) were identified as indicated by arrows.
For comparison, the nucleotide sequences of the rat and human HGF
promoter element are also presented. The functional binding sites for
NF1, USF, AP2, and an E box (i.e. USF binding site) are also
shown. C, mutated oligonucleotides M1-M5 (for sequence
information, please see E) were synthesized and used as
competitors as described under A. D, the S2
oligonucleotide was labeled and used as a probe with NIH3T3 nuclear
extracts to further define the IR2 site. The nuclear extract used in
this gel shift assay was not depleted of NF1. The DR1 site (direct
AGGTCA motif with one spacing) was also used as competitor.
E, the nucleotide sequences of the wild-type and mutant
versions of the promoter element used to define the PPAR binding
site are shown. The mutated nucleotides are underlined.
C2 denotes complex C2. Self-competitor (wild-type unlabeled
probe) was used as a positive control.
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Fig. 3.
Identification of the nuclear receptor as
PPAR and its binding motif as an IR2.
Supershift assays of complex C2 bound to the HGF promoter regulatory
element using antibodies against various nuclear receptors as indicated
in the figure. NF1-depleted nuclear protein extracts from the NIH3T3
fibroblasts and 3T3-L1 fibroblasts were used as indicated in the
figure. C1 denotes complex C1; C2 denotes complex
C2; F denotes free probe; S denotes supershift
complex.
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Regulatory Function of PPAR
and Its Ligand 15-Deoxy-PGJ2 on the
HGF Gene Promoter Activity--
To demonstrate whether the PPAR
binding site and its binding transcription factor PPAR
have
regulatory function on the HGF gene promoter, we performed
cotransfection experiments with HGF-CAT promoter constructs and
PPAR
1 expression vector. Because the PPAR
binding site overlaps
with those of NF1 and USF and because we have shown that NF1 strongly
suppresses HGF gene promoter activity (21), we decided to eliminate the
interference of NF1 with the PPAR
function. To do this, two
different HGF-CAT constructs were generated and used: the wild-type 0.7 HGF-CAT construct and its mutated version, the 0.7 HGF-CAT/NF1-M
construct, in which the NF1 site is mutated but the PPAR
binding
site remains intact. Another HGF-CAT construct, 0.1 HGF-CAT, which does
not have the NF1 and PPAR
binding sites, was also used as a negative
control. We selected the 3T3-L1 cell line because the nuclear protein
extract from this cell line did not form a USF complex (C1) (see Figs. 1 and 3) with this element that may interfere with the PPAR
function. As shown in Fig. 4,
15-deoxy-PGJ2, the PPAR
ligand, significantly induced the promoter
activities of both the 0.7 HGF-CAT and the 0.7 HGF-CAT/NF1-M constructs
but not the 0.1 HGF-CAT construct (Fig. 4). Overexpression of PPAR
1
and treatment with its ligand, 15-deoxy-PGJ2, had an even stronger
stimulatory effect on both the 0.7 HGF-CAT and the 0.7 HGF-CAT/NF1-M
constructs (Fig. 4). It is believed that RXR
is the functional
partner of PPAR
through heterodimer formation (28). Even though we
did not see an obvious supershift complex by anti-RXR
antibody using
fibroblast nuclear extracts (see Fig. 3), it is possible that the
nuclear protein extracts from NIH3T3 and 3T3-L1 cells do not contain a
sufficient amount of RXR
binding activity. In fact, we show that the
HGF PPAR
element binds to both PPAR
and RXR
using liver
nuclear protein extract, which is presumably rich in RXR
(see
below). To find out whether RXR
can regulate the HGF gene promoter
through binding to this IR2 element as a partner of PPAR
, RXR
expression vector and its ligand, 9-cis-RA, were used in
cotransfection CAT assay experiments using a fibroblast cell line. As
shown in Fig. 4, 9-cis-RA itself had a modest effect, but a combination
of 9-cis-RA plus its receptor, RXR
, strongly stimulated HGF promoter
activity, similar to 15-deoxy-PGJ2 with its receptor, PPAR
. The
strongest induction of the promoter was achieved by cotransfection of
PPAR
and RXR
expression vectors in the presence of 15-deoxy-PGJ2
(the PPAR
ligand) and 9-cis-RA (the RXR
ligand) (Fig. 4). Because PPAR
/RXR
could induce the promoter activity of the 0.7 HGF-CAT/NF1-M construct, these results indicate that PPAR
/RXR
may
not only indirectly (via competing with NF1 binding), but also
directly, stimulate the HGF promoter through binding to a functional
RGGTCA motif in the HGF proximal promoter.

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Fig. 4.
Functional analysis of the
PPAR binding site in the HGF gene
promoter. Wild-type HGF-CAT reporter construct (0.7 HGF-CAT) and
the mutated HGF-CAT reporter plasmid (0.7 HGF-CAT/NF1-M), which harbors
a mutation in the NF1 binding site, were used to determine the function
of PPAR on the activity of the HGF promoter. 3T3-L1 cells were
transiently transfected with the HGF-CAT constructs described above.
For cotransfection, 5 µg of RXR and PPAR 1 expression vectors
per each reaction were added. RXR ligand, 9-cis-RA, and PPAR
ligand, 15-deoxy PGJ2, were also added to activate their cognate
receptors. The 0.1 HGF-CAT construct was used as a negative control.
The data are presented as relative CAT activity (percent conversion)
and are from three independent experiments performed in duplicate.
Error bars represent the standard deviation.
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Binding of the Nuclear Hormone Element with RXR
, PPAR
, and
COUP-TF from Liver Tissue and Related Cells--
It is well known that
the HGF gene is expressed in mesenchymal, but not epithelial, cells of
a given tissue. In the liver, HGF is expressed in the non-parenchymal
mesenchymal cells (especially stellate cells, also known as the Ito
cells or lipocytes because they store lipids and vitamin A). We tested
whether the HGF promoter element could bind to PPAR
and possibly
other transcription factors present in non-parenchymal liver cells.
Nuclear protein extracts from whole liver and cells isolated from
liver, such as freshly isolated non-parenchymal cells, as well as an
Ito cell line (25, 26), were prepared. We then subjected these extracts
to gel mobility shift assay using the HGF promoter composite
site as a probe. The probe bound to PPAR
present in all three
different nuclear protein extracts, as shown by supershift assays (Fig. 5, lanes 2 and 7,
and data not shown for whole liver). A surprising finding in these
experiments was that this element could also strongly bind to COUP-TF,
a member of the orphan nuclear receptor subfamily, which also binds to
the RGGTCA motif (see Fig. 5, lanes 3 and 8)
(28). The fact that anti-COUP-TF totally supershifted the C2 complex
whereas anti-PPAR
partially did so (Fig. 5, compare lanes
2 and 3) suggests that PPAR
and COUP-TF may form
heterodimers on the HGF promoter element. Of course, it is known that
COUP-TF also forms homodimers; thus, it is likely that the C2 complex in the nuclear extract of liver cells is mainly composed of COUP-TF homodimer as well as COUP-TF and PPAR
heterodimers. On the other hand, the C2 complex produced by fibroblast nuclear extracts lacks any
COUP-TF, because it did not react with anti-COUP-TF (data not shown;
also see Fig. 3). Anti-RXR
antibody was also used in these
experiments, because RXR
is believed to be the partner of PPAR
,
as mentioned above. We did detect weak RXR
binding to this element
with the nuclear protein extracts from whole liver tissue (the shifted
band was very faint and required a very long exposure of the film)
(data not shown). Taken together, the results imply that the PPAR
binding site in the HGF gene promoter also binds to COUP-TF and RXR
,
depending on the cell types/tissues analyzed (i.e.
fibroblasts versus liver cells).

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Fig. 5.
The IR2 element in the HGF promoter composite
site binds to the nuclear receptors PPAR and
COUP-TF present in the nuclear protein extracts from liver cells.
Supershift assays were carried out using various antibodies as
indicated and nuclear protein extracts from an Ito cell line derived
from rat liver and from freshly isolated non-parenchymal rat liver
cells (NPC). In this experiment, the HGF promoter composite
element was used as a probe. Unlabeled probe (cold self) was used as a
competitor to confirm specificity. S denotes the
supershifted complex.
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To confirm that COUP-TF truly binds to the HGF proximal promoter
composite element, in vitro translated COUP-TF transcription factor was prepared and used in gel mobility shift assays. Indeed, in vitro translated COUP-TF could specifically and avidly
bind to this region, as shown in Fig. 6,
lane 1. Unlabeled excess probe totally competed for
this complex (Fig. 6, lane 2). Moreover, anti-COUP-TF
antibody reacted with and shifted this complex (Fig. 6, lane
4). On the other hand, anti-PPAR
antibody did not react with
this complex (Fig. 6, lane 3). These results prove that
COUP-TF binds specifically to this region. To define the binding
motif(s) for COUP-TF in this region, gel shift competition assays were performed using the truncated oligonucleotides S1-S3 and the mutated oligonucleotides M1-M5 (see Fig. 2E for sequences). S1
oligonucleotide retained all of its binding activity. S2
oligonucleotide retained some of its binding ability, but the S3
oligonucleotide totally lost its binding ability for COUP-TF (data not
shown). Similarly, the competitive ability of M1, M2, and M3 mutated
oligonucleotides reduced only modestly. However, the binding ability
for COUP-TF was totally abrogated when the AGGTCA motif in the 3' end
of the element was mutated (oligonucleotides M4 and M5; data not
shown). Combining all of the above results, we conclude that COUP-TF
also binds to the composite site in this regulatory region of the HGF gene promoter (see Fig. 9). It should be added that the specific binding of the PPAR
isoforms (PPAR
1 and PPAR
2) to the HGF
promoter element was also confirmed in a series of gel mobility band
shift and super shift assays using in vitro translated mouse
PPAR
1, PPAR
2, and RXR
(data not shown).

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Fig. 6.
Characterization of the COUP-TF binding motif
in the HGF promoter composite element. In vitro
translated COUP-TFI protein was used in these experiments. Supershift
assays were performed with antibody against COUP-TF and the wild-type
HGF promoter element as a probe. Self-competitor was used to show the
specific binding of COUP-TFI with this element. Antibody against
PPAR was used as a negative control. The COUP-TF1 complex is
indicated by an arrow. S denotes the supershift
complex. Negative controls for the in vitro translation
product (empty vector control subjected to the in vitro
translation reaction) and gel shift assays were also carried out to
also ensure specificity (data not shown).
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Modulation of PPAR
Function on the HGF Gene Promoter by COUP-TF,
NF1, and AP2--
Because our data showed that COUP-TF binds to the
same binding site as that for PPAR
, it is reasonable to
suppose that COUP-TF homodimers could modify the stimulatory
function of PPAR
/RXR
by competing with one another for their
common binding site. Additionally, published data demonstrate that
COUP-TF can form heterodimers with RXR
in solution in the absence of
their binding site. In this manner, COUP-TF sequesters RXR
,
preventing PPAR
/RXR
heterodimer formation and the stimulatory
function of PPAR
/RXR
. Furthermore, the binding sites for COUP-TF
and PPAR
/RXR
overlap with the binding sites for the suppressive
transcription factors NF1 and AP2. Therefore, the functionality of this
unique composite element to regulate HGF gene expression in a given
tissue or cell may be dependent on the specific combination, and/or the
various concentrations, of transcription factors. To show the
individual functions of these transcription factors and how they
interact to exert their effects, we used the 0.3 HGF-CAT promoter
construct, which has a basal promoter and the proximal region of the
HGF promoter having the composite site containing the NF1, AP2, and IR2
binding motifs. We also used the 0.1 HGF-CAT construct as a negative
control, because it only contains the basal promoter. As shown in Fig. 7, COUP-TF dose-dependently
repressed the stimulatory function of PPAR
/RXR
on the HGF gene
promoter in the 0.3 HGF-CAT construct. It did not have significant
repressive function on the 0.1 HGF-CAT construct (Fig. 7). In
similar experiments using the expression vectors for the NF1/X isoform
and AP2 co-transfected with HGF-CAT constructs we found that they also
inhibited the stimulatory function of PPAR
/RXR
on the HGF gene
promoter in the 0.3 HGF-CAT construct but not in the 0.1 HGF-CAT
construct (data not shown). In these experiments, empty vector controls
did not have any effect on the activity of either HGF-CAT construct
(data not shown). These experiments indicate that NF1, AP2, and
COUP-TFI could individually down-regulate the stimulatory function of
PPAR
/RXR
on HGF gene expression and that they may do this through
competing with PPAR
/RXR
for the overlapping composite binding
site in the HGF proximal promoter region.

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Fig. 7.
Repression of the stimulatory function of
PPAR by COUP-TFI on the HGF gene
promoter. Five µg each of 0.3 and 0.1 HGF-CAT reporter plasmids
was cotransfected along with 2 µg of PPAR 1, 2 µg of RXR , and
increasing amounts of COUP-TFI expression vector into the 3T3-L1
fibroblast cell line. The activity of each promoter construct was
determined and is expressed as the relative CAT activity normalized by
-galactosidase. The results are from three independent experiments
performed in duplicate. Error bars represent the standard error of the
mean.
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Induction of Endogenous HGF mRNA and Protein Expression
by PPAR
Ligand, 15-Deoxy-PGJ2--
To determine whether 15-deoxy
PGJ2 can induce endogenous HGF gene expression at the mRNA and
protein levels, we treated fibroblasts, which naturally express
PPAR
, with 15-deoxy PGJ2 and determined the level of HGF mRNA by
semi-quantitative RT-PCR and the level of HGF protein by a very
sensitive sandwich enzyme-linked immunosorbent assay. The results
demonstrated that HGF expression is significantly up-regulated after
treatment with 15-deoxy-PGJ2 (Fig. 8, A
and B, respectively).

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Fig. 8.
Induction of endogenous HGF mRNA and
protein by PPAR ligand, 15-deoxy PGJ2, in
fibroblasts. A, total mRNA was isolated from 3T3-L1
cells that had been treated with 15-deoxy PGJ2 for different lengths of
time as indicated. Semi-quantitative RT-PCR was carried out as
described under "Materials and Methods." The size of the internal
control -actin is 870 bp (bottom panel). RT-PCR for HGF
yields a 300-bp HGF product (top panel). The left
lane is the molecular weight marker X174/HaeIII.
B, MRC-5 human fibroblasts were cultured in 6-well plates
and treated with or without (control) 15-deoxy PGJ2 in duplicates.
Culture medium was collected at the indicated time points and then
subjected to a sensitive sandwich enzyme-linked immunosorbent assay
using the R&D HGF enzyme-linked immunosorbent assay kit. The amounts of
HGF were determined in the culture medium from the standard curve using
pure HGF as a standard as recommended by the supplier.
Asterisks indicate statistically significant differences
between the control and treated samples (p < 0.05).
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DISCUSSION |
In this study, we demonstrated that the nuclear receptors PPAR
and COUP-TF bind to a composite regulatory element in the HGF upstream
promoter region located at
246 to
233 bp from the transcription
start site. This binding site also overlaps/harbors functional binding
sites for NF1, AP2, and the USF family of transcription factors (21,
22), and together, they constitute a multifunctional composite site to
which these various transcription factors can bind and regulate the
activity of the HGF promoter. Cotransfection assays showed that
PPAR
1, with its partner, RXR
, in the presence of their
corresponding ligands, 15-deoxy PGJ2 and 9-cis-RA, respectively, strongly stimulated HGF promoter activity. NF1, AP2, and COUP-TFI suppressed the stimulatory function of PPAR
by competing for their
individual binding sites. Moreover, we demonstrated that 15-deoxy-PGJ2,
the ligand of PPAR
, induces the expression of the endogenous HGF
gene at the mRNA and protein levels in cultured fibroblasts.
PPAR
is a member of the nuclear receptor superfamily that includes
receptors for the steroid, thyroid, and retinoid hormones (23, 28). It
is known that there are three related but quite distinct PPAR proteins:
PPAR
, PPAR
, and PPAR
. Two forms of PPAR
,
1 and
2,
exist as products of alternative promoter usage (29, 30). Like other
members of this superfamily, PPAR
contains a central DNA binding
domain that binds to a cis-acting element in the promoter of
its target genes. Most of the PPAR
response elements described are
composed of a directly repeating core site separated by one nucleotide
(RGGTCA-N-RGGTCA) called DR1 (24, 28). Interestingly, the PPAR
response element in the HGF promoter region is an inverted repeating
core site separated by two nucleotides (GGGCCA-NN-TGACCT) that we have
called IR2. Published data indicate that PPAR
heterodimerizes with
RXR
and that the PAR
/RXR
heterodimer binds to its response
element and activates its target genes (23, 31). Our results indicated
that the IR2 site in the HGF gene promoter also binds to RXR
, though
very weakly. Generally, RXR
is said to be a silent partner.
Nonetheless, there are several examples in which RXR
can be an
active partner (31). Similarly, both RXR
and PPAR
in the
RXR
/PPAR
heterodimers bound to the IR2 element in HGF gene
promoter region are independently responsive and are synergistically
activated in the presence of both ligands, 15-deoxy PGJ2 and
9-cis-RA.
PPAR
plays an essential role in cell growth and differentiation as
well as in oncogenesis. Controversies exist on whether PPAR
acts as
a tumor promoter or tumor suppressor. For example, ligand activation of
PPAR
causes most, but not all, colon cancer cell lines to undergo a
differentiating response and reverse their malignant phenotype
(32). Paradoxically, ligand activation of PPAR
in min
mice, an animal model for familial adenomatous polyposis, seemed to
cause increased polyposis (33, 34). It is demonstrated that
loss-of-function mutations in PPAR
are also
associated with human colon cancer (35). It is not clear
whether this association between colon cancer and PPAR
is involved
in HGF expression in colon cancer tissue, because HGF expression is
also aberrant in colon cancer tissues. Additionally, HGF can also
function paradoxically as a tumor suppressor and tumor promoter in
different settings (2, 3). Little is known about the target genes
activated by PPAR
; our present findings shed significant light on
the matter in the sense that HGF may mediate some of the functions of
PPAR
in tissue growth and differentiation and in cancer.
Like HGF, PPAR
is expressed in various tissues, but predominantly in
the mesenchymal cells, namely adipocytes/fibroblasts (36).
Interestingly, in the liver, HGF is expressed in a specialized fibroblastic cell type called Ito cells, which are also known as
lipocytes (because they store lipids and vitamin A). Like HGF, PPAR
is also expressed in the placenta, and its deficiency leads to
placental dysfunction and death by E10 (37). A qualitatively similar
placental abnormality has been seen in RXR
knockout mice, suggesting
that these phenotypes may be due to impaired PPAR
-mediated signaling
in the placenta (37). Interestingly, HGF knockout mice revealed that
homozygous HGF mutant embryos have severely impaired placentas with
markedly reduced numbers of labyrinthine trophoblast cells, and the
embryos die before birth by E15 (6, 7). Combining these data, it seems
possible that HGF may be one of the mediators of the growth regulatory
action of PPAR
/RXR
.
So far, we have identified several functional regulatory elements
and their binding transcription factors that act on the HGF gene
promoter (Fig. 9). Based on the current
findings and previous published results, we believe that
transcriptional regulation of the HGF gene is very complex and is
dependent on the combination of all of these transcription factors,
including COUP-TF, CCAAT/enhancer-binding protein, Sp1,
estrogen receptor, NF1, AP2, USF, and PPAR
(15-22). CCAAT/enhancer-binding protein
, SP1, estrogen receptor, USF, and
PPAR
are positive regulators. COUP-TF, AP2, and NF1 are mainly negative regulators. COUP-TFs can compete with both estrogen
receptor and PPAR
for their respective binding sites and
suppress the stimulatory functions of both estrogen receptor and
PPAR
in the presence of their own ligands (estrogen and 15-deoxy
PGJ2, respectively). This result implies that COUP-TFs and the NF1
family play an important role in regulation of HGF gene expression. As
mentioned above, PPAR
and CCAAT/enhancer-binding protein
are believed to play an important role in adipocyte differentiation
(38). Interestingly, we noted that in the 3T3-L1 fibroblasts that
differentiate into adipocytes when placed in differentiation medium, a
marked increase in PPAR
and CCAAT/enhancer-binding protein
binding to the HGF promoter elements occurred and is depicted in Fig.
9. The increase in the binding activity correlated well with an
increase in HGF and HGF receptor (Met) expression in these cells,
implying that the HGF/Met signaling system may have a role in adipocyte
differentiation. A summary of the positive and negative regulators of
the HGF gene promoter that we have identified and characterized thus
far is presented in Fig. 9.

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Fig. 9.
A diagram depicting several functional
cis-acting elements and their cognate transcription
factors within the murine HGF promoter region. NHR,
nuclear hormone receptor; Rep, an unknown repressor;
GTF, general transcription factors;
C/EBP , CCAAT; IL-1, interleukin 1;
TNF , tumor necrosis factor ; EGF,
enhancer-binding protein .
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