From the Department of Pathology, Division of Cellular and
Molecular Pathology, University of Pittsburgh, School of Medicine,
Pittsburgh, Pennsylvania 15261
To understand the molecular mechanisms of
hepatocyte growth factor (HGF) gene transcription in vivo,
we report the generation and characterization of transgenic mice
harboring various lengths of the mouse HGF promoter linked to the
chloramphenicol acetyltransferase reporter gene. Analysis of different
tissues of the transgenic mouse lines having the 2.7-kilobase (kb)
promoter construct revealed a pattern of reporter gene expression in
embryonic and adult tissues that paralleled that of endogenous HGF gene
expression. A similar expression pattern was observed in the 0.7-kb
transgenic lines. However, in contrast to in vitro data, no
promoter activity was detected in four independent transgenic lines
harboring the 0.1-kb construct. Akin to the activity of the endogenous
HGF gene, which is induced in the liver, lung, and spleen in response
to 70% partial hepatectomy, the reporter gene driven by the 2.7-kb
promoter construct was strongly induced, whereas that driven by the
0.7-kb promoter construct was modestly induced in these organs after
partial hepatectomy. Together, these data suggest that the region
between
0.1 and
0.7 kb of the HGF gene promoter is essential to
drive its expression in vivo and that additional upstream
sequences located between
0.7 and
2.7 kb are also necessary for its
maximum inducibility in response to cues that stimulate tissue growth
and regeneration.
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INTRODUCTION |
Hepatocyte growth factor
(HGF)1 is a unique secreted
regulatory molecule with multiple biological activities. It is a strong mitogen for various epithelial cells such as hepatocytes as well as
endothelial cells and melanocytes (1-6) and has been shown to have
motogenic, morphogenic, and antitumor activities (7-11). It exerts
these diverse activities through its specific transmembrane tyrosine
kinase receptor c-Met, also called the HGF receptor (12-14). HGF gene
expression is mainly confined to the mesenchymal/stromal cells of a
variety of tissues under normal and pathophysiological conditions (15,
16). The HGF receptor, on the other hand, is expressed predominantly in
epithelial cells (17, 18). This ligand/receptor system is thought
to play a significant role in mediating stromal-epithelial
interactions (5, 6).
Studies have shown that the HGF gene is expressed in developing embryos
(19), whereas gene knockout studies have revealed that HGF is essential
for normal development of liver, placenta, and muscle (20, 21). HGF
gene expression has been shown to be modulated in vitro by
estrogen, dexamethasone, transforming growth factor-
, tumor necrosis
factor-
, and other cytokines (29-31), and it increases dramatically
in vivo in remnant tissues and distal organs following
tissue loss such as partial hepatectomy, unilateral nephrectomy, or
acute lung injury (22, 23, 26, 32). Thus, it follows that HGF and the
HGF receptor are implicated as an important paracrine system involved
in embryogenesis, organ regeneration, wound repair, and cancer
(19-28). Unveiling the molecular mechanisms that govern HGF and HGF
receptor expression is crucial to understanding the biology and
pathobiology of each of these processes.
In an effort to elucidate the molecular mechanisms responsible for the
complex regulation of HGF gene expression, we have previously reported
the cloning and partial characterization of the 5'-flanking region of
the mouse HGF gene (33). Transient transfection studies using in
vitro models have identified several regulatory elements that may
be involved in the cell type-specific and inducible expression of the
HGF gene (29, 34-38). Promoter analysis using in vitro
transfection assays, however, suffers from many shortcomings and is
limited to few cell types and experiments. Thus, to expand our in
vitro findings as well as to further characterize other mechanisms
involved in regulation of HGF expression in vivo, we
developed transgenic mice harboring chimeric genes containing various
lengths of the mouse HGF promoter region fused to the chloramphenicol
acetyltransferase (CAT) reporter gene. Our data show that the upstream
regulatory regions of the HGF promoter are essential for efficient
promoter activity and contain the necessary DNA elements to dictate
expression in embryonic and adult tissues and to confer inducibility in
response to cues such as tissue loss, which triggers cell growth and
regeneration. These transgenic mice provide a good model system for
studying the transcriptional regulation of HGF gene expression in
normal development and in pathological conditions such as cancer in
which HGF gene expression is unregulated.
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EXPERIMENTAL PROCEDURES |
Generation and Identification of Transgenic Mice--
The
cloning of the mouse HGF promoter region into pCAT basic vector
designated 2.7HGF-CAT (-2674 to +29 bp), 0.7HGF-CAT (
699 to +29 bp),
and 0.1HGF-CAT (
70 to +29 bp) has been described in our previous
study (33). The transgene constructs used for microinjection were
derived from these plasmids by digesting them with HindIII
and HpaI to release the corresponding fragment, which consisted of the HGF promoter sequences, the CAT gene, and the late
SV40 poly(A) site as depicted in Fig. 1. These linearized DNA fragments
were separated by agarose gel electrophoresis and purified by glassmilk
(BIO 101, Inc., Vista, CA). Transgenic mice were generated according to
established methods (39) by the transgenic mouse facility at our
institution using the mouse strain B6D2 (C57B6 × DBA2). Potential
founders were screened for transgene integration by PCR analysis of
blood DNA using CAT-specific primers. Whole blood (50 µl) was
obtained from 3-week-old mice by retro-orbital bleeding, and the DNA
was prepared according to Innis et al. (40). Oligonucleotide
primers designed from the CAT gene sequence (sense, 5'-CACCGTTGATATATCCCAATGGCAT-3'; and antisense,
5'-GCCACTCATCGCAGTAACTGTTGTAA-3') were used to generate a 620-bp PCR
product from the transgene. PCR was carried out using Taq
DNA polymerase (Boehringer Mannheim), 1 µg of blood DNA, and 15 pmol
of primers according to the manufacturer's instructions.
Southern blot hybridization of tail DNA was also performed to
confirm the PCR results. Tails were clipped at postnatal day 21, and
the genomic DNA was extracted according to Hogan et al. (39). Tail DNA (20 µg) was digested with EcoRI restriction
endonuclease, fractionated on 0.8% agarose gel, transferred to
GeneScreen Plus membranes (NEN Life Science Products), and hybridized
with a 32P-labeled 0.6-kb CAT DNA fragment. Hybridization
conditions have been described (41).
CAT Assay--
To determine the expression of the HGF
promoter-CAT reporter constructs in various tissues, CAT activity was
analyzed as follows. Tissues were dissected and homogenized in 0.25 M Tris-HCl (pH 7.5) using a Dounce homogenizer. For cell
suspensions, cells were pelleted, and their membranes were disrupted
using three freeze-thaw cycles. The homogenates were incubated at
65 °C for 5 min and then centrifuged at 15,000 × g
for 10 min at 4 °C. The protein concentration was determined using a
protein assay kit (Bio-Rad). CAT activity was determined by incubating
20-100 µg of total protein with [14C]chloramphenicol
(Amersham Pharmacia Biotech) as a substrate for 24 h as described
(42). The acetylated products were separated by thin-layer
chromatography (Eastman Kodak Co.) and visualized by autoradiography.
Densitometric analysis of autoradiographs was performed using the
BioImage analytical scanning densitometer (Millipore/BioImage, Bedford,
MA) in conjunction with the Whole Band Analysis software package. CAT
activity (percent conversion of CAT substrate to acetylated products)
was normalized by comparison with protein concentration.
RNA Isolation and Analysis
--
Various tissues were dissected
from both wild-type and HGF-CAT transgenic mice anesthetized with
methoxyflurane (Pittman-Moore, Mundelein, IL). Total RNA was then
isolated using RNAzol B solution (Cinna/Biotecx, Friendswood, TX)
according to the manufacturer's instructions. RNA concentration was
determined by measuring the optical density at 260 nm. Northern blots
were prepared by separating 20 µg of total RNA on
formaldehyde-containing 1.0% agarose gels and transferring them to
GeneScreen Plus membranes. Blots were hybridized with
32P-labeled cDNA probes for mouse HGF, CAT, or
glyceraldehyde-3-phosphate dehydrogenase as described (29).
For RT-PCR analysis, 1 µg of total RNA was reverse-transcribed using
avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim)
and amplified with Taq DNA polymerase following the manufacturer's instructions using primers specific to mouse HGF (sense, 5'-ATCAGACACCACACCGGCACAAAT-3'; and antisense,
5'-GAAATAGGGCAATAATCCCAAGGAA-3') (43), CAT (described above), or
-actin (CLONTECH, Palo Alto, CA).
-Actin
served as an RNA integrity and normalization control. The DNA
amplification reaction was also carried out without reverse transcriptase to ensure that the amplified bands were not due to
contamination by genomic DNA in the RNA preparations.
Cell Isolation/Separation--
Hepatocytes and non-parenchymal
liver cells from HGF-CAT transgenic mice and wild-type mice were
isolated by in situ perfusion of the liver with collagenase
(Boehringer Mannheim) as described previously (44). Hepatocyte and
non-parenchymal liver cell suspensions were pelleted by centrifugation
and either disrupted by three freeze-thaw cycles for CAT assay or
extracted with RNAzol B for RNA analysis as described above.
Spleens were excised and teased apart in ice-cold minimal essential
medium containing nonessential amino acids (Life Technologies, Inc.).
This suspension was then loaded onto a Ficoll-Paque gradient (Amersham
Pharmacia Biotech) and centrifuged to separate the cells into a
monocyte/lymphocyte-enriched fraction and a polymorphonuclear cell-enriched fraction. Each fraction was washed in minimal essential medium and assayed for CAT activity as described above.
Peritoneal cell exudates were isolated by lavage using a 10-ml syringe
attached to an 18-gauge needle and cold phosphate-buffered saline from
control mice and mice subjected to abdominal incision and closing, such
as that required for hepatectomy. The cells were pelleted, washed by
centrifugation, and used to prepare cell extracts/RNA or cultured
overnight in RPMI 1640 medium containing 10% fetal bovine serum to
obtain adherent and nonadherent cell populations.
Primary Cell Culture--
Fibroblast cell cultures were
established by the explantation method from the skin of 2.7HGF-CAT
transgenic mice. Fibroblast cultures were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
antibiotics. Cell cultures were passaged by trypsinization and
subjected to study at passage 3 or 4.
Gel Retardation Assays--
A DNA fragment corresponding to
538 to
274 bp of the 5'-flanking region of the mouse HGF gene was
isolated from the mouse chimeric plasmid 0.7HGF-CAT. Following
restriction digestion of mouse 0.7HGF-CAT with SacI and
SmaI, the 264-bp fragment was purified and labeled with
[
-32P]dATP (6000 Ci/mmol; Amersham Pharmacia Biotech)
using T4 kinase (Life Technologies, Inc.). The labeled probe was then
gel-purified and used in gel mobility shift assays as described
previously (36). One µg of poly(dI-dC) (Amersham Pharmacia Biotech)
was used as the nonspecific competitor in 10 µl of reaction mixture. The binding reactions were carried out at room temperature for 20 min
before loading onto 5% nondenaturing polyacrylamide gels (19:1
acrylamide/bisacrylamide). Nuclear protein extracts from liver tissue
and HL-60 cells were prepared as described (34). The concentration of
nuclear extract used in each reaction was 4 µg, and that of the
labeled probe was between 0.2 and 0.4 ng. The same nuclear extracts
were subjected to gel shift assays using a radiolabeled Sp1 site as a
probe to control for integrity of the nuclear protein extracts
described previously (37). For competition experiments, a 100-fold
molar excess of unlabeled DNA fragment was included in the reaction
mixture. Gels were run in 0.5× 0.045 M Tris borate and
0.001 M EDTA at a constant voltage of 190 V, dried, and
autoradiographed with intensifying screens.
DNase I-hypersensitive Site Analysis--
Lung and liver tissues
were dissected from control and 70% partially hepatectomized mice;
rinsed in ice-cold phosphate-buffered saline; and homogenized in 2 ml
of 0.2% Nonidet P-40, 60 mM KCl, 15 mM NaCl,
0.05 mM CaCl2, 3 mM
MgCl2, 0.5 mM dithiothreitol, 250 mM sucrose, and 15 mM Tris-Cl (pH 7.4) by 20 strokes in a Dounce homogenizer. Aliquots of 400 µl were then treated
with DNase I (0-30 units; Boehringer Mannheim) for 1 min at room
temperature. The reaction was terminated by the addition of EDTA and
SDS to final concentrations of 12.5 mM and 0.5%,
respectively. DNA was isolated by phenol extraction following a 30-min
RNase A digestion. DNA was then digested with
HindIII/EcoRI, run on 1.5% agarose gel,
transferred to GeneScreen Plus membranes, hybridized with a DNA
fragment that corresponded to the 5'-flanking region of the mouse HGF
promoter, and autoradiographed.
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RESULTS |
Development of the Chimeric HGF Promoter-CAT Transgenic
Mice--
We generated transgenic mouse lines that harbor various
lengths of the mouse HGF promoter region (5'-deletions) fused to the CAT reporter gene. The constructs designated 2.7HGF-CAT (
2674 to +29
bp), 0.7HGF-CAT (
699 to +29 bp), and 0.1HGF-CAT (
70 to +29 bp)
(Fig. 1) were used since our previous
in vitro transient transfection studies using these
constructs revealed that they are transcriptionally active and may
contain important regulatory elements (33, 34, 36). Transgenic
offspring were screened for germ line integration of the transgene by
both PCR amplification of peripheral blood cell DNA and Southern blot
hybridization of tail DNA (data not shown). Four lines with germ line
integration of the 2.7HGF-CAT transgene construct were propagated. Of
these, two lines contained ~10 copies of the transgene, whereas the
other two had five copies. Three transgenic founders harboring the
0.7HGF-CAT construct were also established. Estimates of transgene copy
numbers indicated that all founders possessing the 0.7HGF-CAT construct contained approximately five copies; however, one founder did not yield
germ line transmission. Germ line transmission of the 0.1HGF-CAT
construct was propagated in four transgenic lines, two of which had
approximately five copies and two of which harbored 10-15 copies of
the transgene (data not shown).

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Fig. 1.
Schematic representation of HGF promoter-CAT
reporter gene constructs and identification of the transgenic
mice. Shown is a diagram depicting the three 5'-deletion chimeric
HGF promoter-CAT constructs containing 2.7, 0.7, and 0.1 kb of the
mouse HGF promoter linked to the 0.7-kb CAT coding region and a 0.8-kb
poly(A) site from SV40. Arrows indicate the transcription
initiation site (position +1) of the HGF gene. Drawing is not to
scale.
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Tissue Distribution of HGF-CAT Transgene Expression--
To
determine whether the HGF promoter fragments described above were
transcriptionally active in mice, extracts from various tissues of
HGF-CAT transgenic animals (8-10 weeks of age) were isolated and
assayed for reporter gene expression by CAT assay to determine CAT
activity and by Northern blot analysis to determine CAT mRNA
levels. Tissues from nontransgenic mice were also harvested and
analyzed for expression of the endogenous HGF gene by RT-PCR and
Northern blot analysis. In the 2.7HGF-CAT mice, we observed a
widespread tissue distribution of the reporter gene expression, which
mimicked that of the endogenous HGF gene, with nearly all tissues
examined expressing a detectable level of CAT activity (Fig.
2 and Table
I). A similar expression pattern was
observed with the 0.7HGF-CAT transgenic lines, although the expression in the 2.7HGF-CAT transgenic lines was much more robust than that observed with the 0.7HGF-CAT animals, particularly in the kidney, stomach, and intestine (Table I). Interestingly, the 0.1 HGF-CAT transgenic mice did not express the transgene in any tissues assayed from the four independent founder lines (Fig.
3 and Table I). Akin to the expression of
the endogenous HGF gene, the highest expression of the transgene was
reproducibly noted in the lung, skin, and spleen, especially in the
2.7HGF-CAT mice (Figs. 2 and 3 and Table I). To substantiate these
findings, we analyzed the expression pattern of the endogenous mouse
HGF gene in wild-type littermates by RT-PCR and Northern blot analysis
(Table I). Tissues from nontransgenic littermates were also screened
for CAT activity, and none was detected in any of their extracts, as
expected (Fig. 3).

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Fig. 2.
Tissue distribution of 2.7HGF-CAT construct
expression in transgenic mice. Shown is a graphic representation
of several CAT assay experiments using 50 µg of protein extracted
from various tissues of 10-week-old 2.7HGF-CAT transgenic mice. The
results are presented as mean CAT activity (percent conversion) ± S.D. from at least four independent experiments. Three out of four independent 2.7HGF-CAT lines expressed the transgene in various tissues.
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Table I
Summary of endogenous HGF gene expression in wild-type mouse tissues
and HGF promoter activity in HGF-CAT transgenic mouse tissues
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Fig. 3.
Lack of transgene expression in the
0.1HGF-CAT transgenic mice. Shown are the results from a
representative CAT assay demonstrating the expression pattern of CAT
transgene levels in three separate HGF-CAT transgene constructs
(i.e. 2.7, 0.7, and 0.1 kb). Protein extract (50 µg) from
various tissues of 0.1HGF-CAT, 0.7HGF-CAT, and 2.7HGF-CAT transgenic
mice and nontransgenic (NTG) mice was analyzed for CAT
expression (see Table I). Representative results of CAT assays are
shown for skin, spleen, and lung since these tissues reproducibly
exhibited the highest CAT expression as compared with the other
tissues.
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Cellular Compartments of HGF-CAT Expression--
It is well known
that HGF gene expression is mainly confined to mesenchymally derived
cells of various tissues. For example, in the liver, the HGF gene is
expressed in non-parenchymal cells such as Ito cells, Kupffer cells,
and endothelial cells, but not in parenchymal hepatocytes. To define
the cellular localization of transgene expression, specific cell types
were isolated from HGF-CAT transgenic mice and assayed for CAT
expression. Similar to endogenous HGF gene transcription, transgene
expression (2.7HGF-CAT and 0.7HGF-CAT constructs) was directed to the
non-parenchymal liver cell fraction, but not to the hepatocyte fraction
of the liver (Table II).
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Table II
Summary of endogenous HGF gene expression and HGF promoter activity in
specific cell types isolated from wild-type and HGF-CAT transgenic mice
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Previous studies have shown that peripheral blood leukocytes, human
promyelocytic leukemia cells (HL-60 cell line), and human promonocytic
leukemia cells (THP-1 cell line) express the HGF gene and that the
expression of HGF in these cells is inducible by
12-O-tetradecanoylphorbol-13-acetate and cytokines such as tumor necrosis factor-
(45, 46). High levels of HGF mRNA and CAT
mRNA were detected in both the monocyte- and polymorphonuclear cell-enriched fractions of the spleens of 2.7HGF-CAT and 0.7HGF-CAT transgenic mice (Table II). The spleen mononuclear cell fraction exhibited a significantly higher level of expression of the HGF-CAT transgenes than the polymorphonuclear cell-enriched fraction. Cells
isolated by peritoneal lavage and lung lavage also had detectable levels of both endogenous HGF mRNA and CAT mRNA in both
2.7HGF-CAT and 0.7HGF-CAT transgenic mice (Table II).
Since it is well known that skin fibroblasts express the endogenous HGF
gene, they were isolated from the 2.7HGF-CAT transgenic mice, cultured,
and assayed for CAT activity. CAT assays revealed that these
fibroblasts clearly express detectable levels of CAT activity (Table
II). In addition, these cultures maintained transgene expression when
they were propagated and subcultured up to four passages over the
period of 1 month, suggesting that these cells may provide a unique
system for studying the transcriptional regulation of the mouse HGF
gene under conditions of stable transfection.
Developmentally Dependent Expression of the HGF Promoter--
HGF
mRNA and protein are expressed during embryogenesis (17-19), and
the proper development of organs such as the liver and placenta is
dependent on HGF (20, 21). To examine whether the 2.7-kb fragment of
the HGF promoter region is sufficient to dictate HGF promoter activity
during embryogenesis, CAT activity was measured in tissues isolated
from 2.7HGF-CAT transgenic fetuses at different stages of development.
Similar to the endogenous gene, the promoter construct was active in
embryonic tissues such as placenta, liver, and lung (gestational days
15 and 19 were examined). Endogenous HGF gene expression in lung and
liver tissues increases in the latter part of gestation until
adulthood, when it plateaus in the lung and declines in the liver (17,
19). Similarly, we found that the expression of the 2.7HGF-CAT
construct was readily detectable in embryonic tissues at day 15 (the
earliest time examined) and continued to increase through fetal (day
19) and neonatal (day 3 after birth) development until adulthood, where
it stabilized (Fig. 4). These results
indicate that the 2.7-kb 5'-flanking region of the HGF promoter is
sufficient to dictate proper HGF expression during development.

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Fig. 4.
Expression of the HGF-CAT promoter construct
in tissues of transgenic mice at various stages of development.
Different tissues of the 2.7HGF-CAT transgenic mice harvested during
embryonic and postnatal development were subjected to CAT assays as
described under "Experimental Procedures." The results are
presented as mean CAT activity (percent conversion) ± S.D. from two
independent experiments.
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Induction of the HGF Promoter in Regenerating Liver in Response to
Partial Hepatectomy--
HGF expression in normal adult liver is very
low, but increases in response to loss of hepatic tissue such as
partial hepatectomy (PHX) or hepatotoxin treatment in rats (4, 22, 23).
We first determined that this induction phenomenon also occurs in wild-type mouse liver after partial hepatectomy. As shown in Fig. 5A, HGF mRNA was barely
detectable in the livers of unoperated control animals, but was induced
from 6 to 12 h post-hepatectomy. We then asked whether the HGF
promoter fragments we have cloned contain the necessary element(s) to
confer inducibility in response to cues for tissue regeneration. Thus,
we compared the pattern of CAT expression in various tissues of the
HGF-CAT transgenic mice following PHX. Fig. 5B shows that
the CAT activity in the liver at time 0 (liver tissue removed at the
time of operation from each individual animal; hence, each regenerating
liver has its own time 0 control) is low and is substantially induced
in the corresponding remnant liver at various times post-PHX, peaking at ~24 h post-operation. These phenomena were repeatedly observed in
both 2.7HGF-CAT and 0.7HGF-CAT transgenic mice. Therefore, regulatory
elements responsible for the induced HGF promoter activity in the liver
in response to 70% hepatectomy are apparently contained within the
0.7-kb fragment of the HGF promoter region. However, the inducible
response is stronger in the 2.7-kb construct, and therefore, other
elements between 0.7 and 2.7 kb appear to be necessary for optimal
injury-induced expression.

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Fig. 5.
Induction of the HGF-CAT transgene in the
liver during liver regeneration stimulated by 70% partial hepatectomy.
A, Northern blot analysis showing the induction of
endogenous HGF mRNA in regenerating livers of wild-type mice
post-partial hepatectomy. Total RNA was isolated from mouse liver
tissues at 0, 1, 3, 6, and 12 h after partial hepatectomy;
blotted; and probed with mouse HGF and subsequently with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs,
respectively. B, induction of the HGF-CAT promoter constructs in regenerating livers of transgenic mice. Transgenic mice
were subjected to 70% partial hepatectomy. Tissue was harvested at 0, 12, 24, and 72 h following surgery and subjected to CAT assay as
described under "Experimental Procedures." The results are
presented as bar graphs of mean CAT activity (percent conversion) ± S.D. from three independent experiments performed using 0.7HGF-CAT and
2.7HGF-CAT transgenic mice. No CAT activity was detected in the livers
of the 0.1HGF-CAT transgenic animals even after PHX. *, values are
statistically significant compared with corresponding time 0 controls
(Student's t test; p 0.05).
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The Upstream Region of the HGF Promoter Is Necessary for Its
Maximal Inducibility in the Lung and Spleen after Partial
Hepatectomy--
It has been well documented by several independent
studies that HGF expression is induced in the spleen and lung following partial hepatectomy (22-24). Based on these findings, it has been suggested that HGF also functions in an endocrine manner to facilitate organ regeneration (1-5). Interestingly, in our HGF-CAT transgenic partial hepatectomy models, we noted a significant activation (~3-fold) of the HGF-CAT transgene in the lungs and spleens of the
2.7HGF-CAT transgenic mice, but only a modest, yet significant increase
(1.5-fold) in these organs from the 0.7HGF-CAT animals (Fig.
6).

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Fig. 6.
Induction of the HGF-CAT transgene in lungs
and spleens of 2.7HGF-CAT and 0.7HGF-CAT transgenic mice stimulated by
70% partial hepatectomy. Tissues were harvested at 0, 12, 24, 48, and 72 h following PHX and subjected to CAT assay as described under "Experimental Procedures." The results are presented as bar
graphs of mean CAT activity (percent conversion) ± S.D. from three
independent experiments performed using 0.7HGF-CAT and 2.7HGF-CAT transgenic mice. Strong induction occurred in the lungs and spleens of
the 2.7HGF-CAT animals, whereas moderate induction was noted in the
0.7HGF-CAT mice. No CAT activity was detected in the lungs or spleens
of the 0.1HGF-CAT lines even after PHX. *, values are statistically
significant compared with corresponding time 0 controls (Student's
t test; p 0.05).
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We also examined HGF promoter activity in another injury response
model. We analyzed cells isolated from the peritonea of wild-type and
transgenic mice by lavage following the simple abdominal incision and
closing used in the partial hepatectomy procedure. The endogenous HGF
mRNA was virtually undetectable in cells obtained from peritoneal
lavage of unoperated wild-type mice. However, cells obtained from
peritoneal lavage of wounded wild-type mice (i.e. those
receiving the incision) exhibited significant levels of HGF mRNA
expression at 24 h post-operation as determined by RT-PCR (Fig.
7A). Similarly, peritoneal
cells from the 2.7HGF-CAT transgenic mice showed markedly induced
expression of the CAT transgene in this injury model (Fig.
7B). When the lavaged cells, which consisted mostly of
macrophages and occasional polymorphonuclear leukocytes, were separated
into adherent and nonadherent cell populations by placing them in
culture, CAT expression was mainly detected in the adherent cell
population, of which macrophages are the majority (Fig. 7B).
Despite the fact that transgene expression was very low (yet
detectable) in the peritoneal cells from the 0.7HGF-CAT transgenic
lines, absolutely no induction was noted following incision and closure
(data not shown).

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Fig. 7.
Injury-induced activation of the 2.7HGF-CAT
construct in the peritoneal cells from transgenic mice after abdominal
incision. A, endogenous HGF expression (using wild-type
mice) is induced following abdominal incision as determined by RT-PCR
using HGF-specific primers. One µg of total RNA isolated from the
lavaged peritoneal cells of control mice (lane 2) and
operated mice at 24 h post-surgery (lane 3) was used in
each RT-PCR. Lane 1 contains the DNA size marker
x174/HindIII. Lane 4 contains the RT-PCR
product using mRNA from liver tissue as a positive control.
Lane 5 contains the negative control (mock) RT-PCR lacking
mRNA. RT-PCR with -actin primers was performed to assure that
there were no differences in the integrity and concentration of RNA in
each sample (data not shown). B, representative CAT assay
showing that the expression of the 2.7 HGF-CAT transgene was
dramatically induced in cells collected by peritoneal lavage following
abdominal incision. Lavaged peritoneal cells from control (unoperated)
and operated transgenic animals were separated into nonadherent and
adherent cell populations by culturing them overnight in a plastic
culture dish. CAT activity was then assayed using 50 µg of protein
extract/reaction.
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Our in vivo data clearly demonstrated that the 2.7-kb
promoter region is sufficient to confer high and inducible expression to tissues such as lung and liver and that this promoter region behaves
in a manner similar to that seen with the endogenous HGF gene promoter.
Because of these findings, we performed DNase I-hypersensitive site
assays using freshly isolated nuclei from lungs and livers of wild-type
mice to define the region(s) in this DNA segment that may bind or be
accessible to transcription factors in vivo. The results
indicated that at least five hypersensitive sites (HSS) are scattered
throughout the 2.7-kb HGF promoter region, which roughly map to
positions at
2.2,
1.5,
1.2,
0.7, and
0.3 kb from the
transcription start site. These HSS were more prominent in the DNA
prepared from the lungs of the operated animals (PHX) as compared with
the unoperated controls (Fig. 8). Similar HSS in the 2.7-kb promoter region were also present in the DNA prepared
from liver nuclei (data not shown). These data are indicative of
protein-DNA interactions and transcriptional activity in the endogenous
HGF promoter in vivo.

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Fig. 8.
DNase I-hypersensitive site analysis of the
HGF promoter following 70% hepatectomy. Freshly isolated lung
nuclei from normal and hepatectomized CD-1 mice were incubated with 0 (lanes 1 and 4), 15 (lanes 2 and
5), or 30 (lanes 3 and 6) units of
DNase I at 25 °C for 1 min. Genomic DNA was extracted,
restriction-digested with EcoRI/HindIII, and
subjected to Southern blot analysis using a radiolabeled fragment of
the mouse HGF promoter region as a probe as described under
"Experimental Procedures." The major band is the 3.5-kb
EcoRI/HindIII HGF promoter genomic fragment. DNase I HSS are labeled HSS 1-5.
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Since the 0.7HGF-CAT transgenic construct responded to PHX in the
liver, lung, and spleen (Figs. 5B and 6) and two
injury-induced HSS were detected in this region of the promoter, we
surmised that some injury-inducible response elements are localized to this 0.7-kb region. Interestingly, our previous in vitro
functional analysis of the 2.7-kb promoter using various 5'- and
internal deletion constructs localized regulatory element(s) to the
538 to
274 bp region, which exerted a strong enhancing effect on the activity of the HGF promoter (33). Thus, we performed gel mobility
band shift assays using this fragment of the 0.7-kb HGF promoter as a
probe and liver nuclear protein extracts prepared from normal and
partially hepatectomized wild-type mice. The probe that corresponded to
the promoter region from
538 to
274 base pairs formed a major
specific complex with liver nuclear extracts, which increased in
intensity at 6 h post-hepatectomy (Fig.
9A). The same nuclear extracts
were subjected to gel shift assays using a radiolabeled Sp1 site as a
probe to control for integrity of the nuclear protein extracts (Fig.
9B). The complexes formed by the Sp1 probe, which are not
induced by PHX, are composed of Sp1 and Sp3 proteins, as we described
previously (37). Computer analysis of this
538 to
274 bp DNA
segment identified potential AP-1, cAMP, nuclear factor-IL-6, and IL-6
response elements. However, except for the slight competition observed
with the nuclear factor-IL-6 site (C/EBP site) and the IL-6 response
element (Fig. 9C), none of the binding sites competed
strongly with the labeled probe for formation of the major binding
complex when they were used as competitors in gel mobility band shift
assays.

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Fig. 9.
Induction of a specific HGF promoter-binding
complex in liver nuclear extract following 70% hepatectomy.
A, electrophoretic mobility band shift assays were performed
in the presence of mouse liver nuclear extracts prepared at different
time points including 0 (control), 3, 6, and 12 h after
PHX using -32P-labeled HGF promoter fragment ( 538 to
274 bp) as a probe (F). Specific binding complexes
(C) are effectively competed by a 100-fold molar excess of
unlabeled probe (+). B, the same nuclear extracts were
subjected to gel shift assays using a radiolabeled Sp1 site as a probe
to control for integrity of the nuclear protein extracts. The binding
complexes (C) formed by the Sp1 probe are composed of Sp1
and Sp3 proteins, as we described previously (37). C, shown
are the results from competition analysis using double-stranded oligonucleotides corresponding to various potential binding sites as
competitors. The assay was performed using nuclear protein extract from
regenerating liver (6 h post-hepatectomy) with the same probe and gel
shift conditions described for A.
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DISCUSSION |
HGF is a pleiotropic growth factor that regulates growth and
regeneration of various tissues under normal conditions as well as
during neoplastic growth. Its gene expression is restricted to
mesenchymally derived cells in a multitude of tissues and is induced in
response to tissue loss to facilitate regeneration. HGF gene expression
is influenced by some cytokines and hormones. The primary mechanism of
regulation of many genes is at the level of transcription and involves
an intricate network of interactions between cis-acting DNA
regulatory elements within the promoter and multiple cognate
trans-acting factors. To understand this regulatory
mechanism regarding HGF, we and others have previously cloned and
partially characterized the 5'-flanking region of the mouse HGF gene
promoter. Through these studies, several putative regulatory elements
that control the cell type-specific and inducible expression of this
gene have been identified by in vitro DNA transfection studies (33-38). Although these experimental results are insightful, their true in vivo significance is not known. In the present
study, we have reported the generation and characterization of
transgenic mice harboring chimeric HGF promoter-CAT reporter genes
(containing 2.7, 0.7, or 0.1 kb of the 5'-flanking region of the mouse
HGF promoter linked to the chloramphenicol acetyltransferase reporter gene) for the purpose of studying the in vivo regulation of
the mouse HGF promoter. CAT assays, RT-PCR, and Northern blot analysis have shown that the 2.7HGF-CAT transgenic mice express the HGF promoter-driven transgene in a manner similar to that observed with the
endogenous HGF gene. The expression is evident in a broad spectrum of
tissues and is restricted to mesenchymally derived cells. The
expression is also developmentally regulated and is induced in response
to injury similar to the endogenous gene.
Previous transient transfection investigations from independent
laboratories showed that the 0.1-kb basal promoter region (
70 to +29
bp) was necessary and sufficient to efficiently drive the CAT reporter
gene in vitro (33, 35). The present study on transgenic mice
revealed that the 0.1-kb promoter region is not sufficient to dictate
efficient expression in vivo (Table I). Thus, it is evident
that other distal elements, in addition to those present within the
first 0.1 kb, are required to enforce HGF expression in
vivo. The functional differences observed between transient
transfected cells and transgenic animals may reflect a difference in
the chromatin structure of the transferred genes. Indeed, in many
cases, genes are regulated correctly in transgenic mice, but
inappropriately after transfection into cells (47-49). Furthermore,
while transfection studies in cell culture models may help define
regulatory elements in proximal promoter regions, they may not be able
to identify the importance of distal enhancers or locus control regions
whose functions are critically dependent on higher order chromatin
structure. Such structures help to approximate upstream sequences with
their cognate basal promoters to mediate proper and maximal gene
activation (50-52). For example, the multiple Sp1 sites identified in
the 2.7-kb 5'-flanking region of the HGF promoter may not only function
as enhancers (37), but may also facilitate loop formation. This higher
order structure may in turn provide additional transcription factors
such as C/EBP, the estrogen receptor, and the chicken ovalbumin
upstream promoter transcription factor access to their binding sites in
the HGF promoter for fine-tuned regulation (36, 38).
In vitro studies of the HGF promoter revealed that the HGF
promoter constructs including 2.7HGF-CAT, 0.7HGF-CAT, and 0.1HGF-CAT are active not only in stromal cells such as NIH3T3 fibroblasts, but
also in epithelial cells such as RL95-2 endometrial carcinoma cells and
HepG2 hepatocellular carcinoma cells, which do not express the HGF gene
(33, 36). Our current data show that the promoter indeed behaves like
the endogenous HGF gene in vivo and that the HGF promoter
constructs (2.7 and 0.7 kb) are not expressed in epithelial cells such
as hepatocytes (Table II). These results again imply that in
vitro data obtained by transient transfection are often misleading
in some cases. The data also suggest that the HGF gene promoter in
epithelial tissues may be inactivated during tissue development by some
mechanisms such as DNA methylation and nucleosome phasing.
In response to certain types of injury and cues that trigger tissue
regeneration, many genes are transcriptionally induced or repressed.
The HGF gene is induced in the liver as well as in more distal sites
such as the lung in response to loss of liver mass induced by PHX
(22-24). We have shown that transgene expression driven by the 2.7- and 0.7-kb HGF promoter fragments is also up-regulated in these tissues
following 70% hepatectomy (Figs. 5 and 6). CAT activity in the liver
increased by 12 h post-hepatectomy, peaked at 24 h, and
remained elevated through several days, during which tissue
regeneration occurred. Northern blot analysis showed that CAT mRNA
levels increased at 6-12 h post-PHX in a manner identical to that seen
with endogenous HGF mRNA expression (data not shown). The
differential in time between detected CAT activity and mRNA expression is consistent with the lag time for protein synthesis and
accumulation.
Our present study indicated that additional response elements lying
between
2.7 and
0.7 kb in the HGF promoter enhance the induction of
the HGF promoter in a tissue- and cell-specific manner. We based this
hypothesis on the following observations. Substantial induction of the
2.7HGF-CAT construct was noted in the lung and spleen as well as the
liver after PHX (Figs. 5 and 6). In contrast, the expression of the
0.7HGF-CAT construct was only modestly up-regulated in the liver, lung,
and spleen in response to PHX. Analysis of another injury response
model revealed that a simple abdominal incision or hepatectomy (which
requires such an incision) resulted in a dramatic activation of HGF
expression in lavaged peritoneal cells by 24-48 h post-operation (Fig.
7B). Surprisingly, only the 2.7HGF-CAT transgenic construct,
but not the 0.7HGF-CAT transgenic construct, was induced in this model.
This again implies that an additional regulatory element(s) between
2.7 and
0.7 kb of the mouse HGF promoter, by itself or in concert
with other element(s), is responsible for the induction of HGF promoter
activity. This claim is supported by the presence of multiple DNase
I-hypersensitive sites within the 2.7-kb HGF promoter fragment (Fig.
8), which map to
2.2,
1.5,
1.2,
0.7, and
0.3 kb of the HGF
5'-flanking promoter region.
We have shown that at
872 to
860 bp in the HGF promoter, an
estrogen response element (5'-GGTCAGAAAGACC-3') is present. We
demonstrated that the chicken ovalbumin upstream promoter transcription factor, a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site, effectively silenced the basal transcriptional activity of the HGF promoter (36).
The estrogen receptor, on the other hand, relieved the repressive
action of the chicken ovalbumin upstream promoter transcription factor,
resulting in the induction of the HGF promoter. Injection of estradiol
stimulated HGF promoter activity in tissues such as mammary gland and
ovary of mice harboring the 2.7-kb region, but not the 0.7-kb region,
of the mouse HGF promoter (36). Furthermore, our laboratory recently
reported that members of the C/EBP family (especially C/EBP
) of
transcription factors bound to a region in the basal HGF promoter (at
+1 bp) and were responsible for the inducibility of the promoter by
cytokines such as IL-6 and tumor necrosis factor-
in NIH3T3 cells
in vitro (38). The activity of binding of C/EBP
and
C/EBP
to this region of the HGF promoter was strongly induced in the
liver after partial hepatectomy (38). The fact that the 0.7-kb
construct, but not the 0.1-kb construct, was active in vivo
and was induced in the liver after PHX suggests that other sites, novel
or known, within the 0.7-kb region are responsible for facilitating
induction of the HGF gene in this scenario. Of course, regions upstream
of 0.7 kb also contributed for maximum inducibility, especially in the
lung, spleen, and peritoneal macrophages. In summary, our present
results demonstrate that HGF promoter-CAT transgenic mice provide a
unique system by which we can analyze the true in vivo
transcriptional regulation of the HGF promoter under various normal and
pathological conditions and further characterize the important
regulatory cis-acting elements and their cognate
transcription factors involved in these processes.
We thank Dr. G. K. Michalopoulos for many valuable discussions and continued enthusiasm
during the course of this work. We also thank Dr. M. C. DeFrances
for critical review of this manuscript.