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INTRODUCTION |
Eukaryotic cells have developed specific signal transduction
pathways to respond to and integrate extracellular stimuli. Three of
these pathways that have been elucidated in eukaryotic cells can be
simplified as follows: 1) raf
MEK1,2
ERK1,2; 2) MEKK1
SEK1
JNK/SAPK; and 3) MEKK1
SEK1
p38 (1). In each of these
kinase cascade pathways, the upstream kinase phosphorylates and
activates its immediate downstream substrate kinase. Extracellular signals are thereby transduced through these cytoplasmic kinase cascades to reach their nuclear targets and regulate gene expression. Recent studies indicate that the raf
ERK1,2 pathway has significant effects on
I(I) collagen gene expression in rat-derived hepatic sinusoidal stellate cells
(HSCs),1 the major effector
cells during the overproduction of collagen which typifies hepatic
fibrogenesis and cirrhosis (2-5). The response elements for the
cascade involved a NF-1 site in the proximal promoter of the collagen
gene, as well as a region within
1620 to
1630 in the distal
promoter of the
I(I) collagen gene (2). The importance of the NF-1
site in regulating collagen gene expression is in agreement with other
studies using non-HSCs (6-9). The effects of JNK activation on
I(I)
collagen gene expression had not been evaluated in HSCs. Activated JNK
can phosphorylate and activate its target protein, JUN, and transduce a
signal from cytoplasm to nucleus (10-13). JNKs are activated by a
variety of stimuli, such as UV irradiation, heat shock, and osmotic
imbalance (14, 15). HSCs have been shown to contain the JNK cascade, which can be activated by fibronectin, interleukins, and tumor necrosis
factor (16). These are classic compounds associated with tissue injury
and may also be present in serum (16). The current report studied the
effects of UV irradiation of HSCs on activation of JNK and on
I(I)
collagen gene expression. It was found that exposure of HSCs to UV
light activated JNK but not ERK1,2, and the activation of JNK by UV
irradiation increased
I(I) collagen mRNA abundance. Further
studies indicated that activated JNK regulated
I(I) collagen gene
expression through a distal GC box located in the 5'-UPS of the
I(I)
collagen gene. This response element was distinct from that utilized by
the ERK1,2 cascade. A 32-kDa protein, designated basic transcription
element-binding protein (BTEB), was found to bind to the GC box. BTEB
DNA binding activity was up-regulated in activated stellate cells. The
potential mechanism(s) utilized by UV-induced activation of JNK to
stimulate
I(I) collagen gene transcription is further discussed.
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MATERIALS AND METHODS |
Cell Culture and Transient Transfection--
Hepatic stellate
cells (HSCs) were isolated from Sprague-Dawley male rats and
subcultured in DMEM supplemented with 10% fetal bovine serum and 10%
newborn calf serum (10/10 DMEM) by previously described methods (17,
18). Experimental manipulations were performed with cells at passages
2-6. Sixty to eighty percent confluent cells in 6-well cell culture
plates were transfected by the LipofectAMINE method following the
protocol provided by the manufacturer (Life Technologies, Inc.).
Equimolar colCAT reporters (1-1.3 µg of DNA) and equal amounts of
dominant negative JUN or empty control plasmid pMNC (2 µg) were used
in each well. For each transfection, 0.2 µg of
-galactosidase
expression plasmid pSV-
-gal (Promega) was co-transfected for
evaluation of transfection efficacy. Each DNA transfection experiment
was performed in triplicate. Each transfection was repeated at least
three times.
Chemicals and UV Irradiation--
Unless specifically noted, all
chemicals were purchased from Fisher or Sigma. Seventy to eighty
percent confluent passaged HSCs were incubated in DMEM with 0.4% fetal
bovine serum for 48 h before exposure to a UV lamp (254 nm, 38 W,
76-cm distance between uncovered plates and the UV lamp) in a tissue
culture hood for 0.2 or 0.4 min. The UV dose was approximately 10 J/m2 (19). Control cells were exposed to regular light in a
tissue culture hood for 0.4 min. Cells were then maintained in the
media with 0.4% serum in a 37 °C incubator for an additional 1 h before total RNA isolations or protein extractions.
RNA Isolation and RNase Protection Assay (RPA)--
Total RNA
was isolated by the TRI-Reagent (Sigma) following the protocol provided
by the manufacturer. Single-stranded RNA probe complimentary to
I(I)
collagen mRNA nucleotides was made from the first exon (1-206) of
the gene via polymerase chain reaction (PCR). Primers for the PCR were
5'-CGGGATCCCGAGCAGACGGGAGTTTCACC-3' (the boldface region
generated a BamHI site) and
5'-TCCCCCGGGGGAGAACTTACTGTCTTCTTGG-3' (the boldface region
generated a SmaI site). The PCR product was then subcloned
into BamHI and SmaI sites in the plasmid
pGEM-3Zf(+) (Promega). The T7 promoter in the plasmid was
used to generate single strand antisense RNA probe. The template for
rat cyclophilin was obtained from Ambion and yields a 103-base pair
protected fragment. The antisense RNA probes were synthesized and
labeled by MAXIscript in vitro transcription kits (Ambion).
The synthesized probes were gel purified. RPA was carried out with RPA
II (Ambion) following the protocol provided by the manufacturer. The
dried gel was exposed to a phosphor imaging system (Phosphor Image SI, Molecular Dynamics, Sunnyvale, CA). The radioactivity in each band was
measured by computer-aided densitometry of the phosphorimage using
IPLab Gel (Signal Analytics Corp.) as described previously (20).
CAT Assay and Transfection Efficacy Normalization--
Cells
were harvested and assayed for CAT activity as described previously
(2). In brief, cells were washed twice and harvested in cold PBS by
cell scrapers. Cells in PBS were lysed by three cycles of freeze-thaw.
After centrifugation for 15 min at high speed at 4 °C, protein
concentrations were determined by QuantiGold (Diversified Biotech).
Protein extracts were heated for 5 min at 65 °C to inactivate any
endogenous acetylases. The protein extracts reacted with 50 µl of CAT
assay mixture at 37 °C for 90 min. The mixture contains 350 µl of
PBS, 110 µl of 10 mM acetyl-CoA, 1.5 ml of
chloramphenicol, and 100 µl of 0.5 mCi/ml
[3H]acetyl-CoA. The reaction mixtures were added to
Econofluor-2 (Packard Inc.) in scintillation vials. The CAT activity
was analyzed in a liquid scintillation analyzer. Transfection efficacy
was normalized by analyzing co-transfected
-galactosidase activity expressed as
-gal relative unit/mg protein by utilizing
Galacto-Light chemiluminescent reporter
-galactosidase assay kit
(Tropix). Finally, the CAT activity of each transfection was expressed
as relative units/mg of protein after normalization of transfection efficacy as calculated by
-galactosidase activity. Transfection efficiency was estimated by counting blue cells as described by Promega. Cells transfected with the
-gal reporter plasmid were stained blue by x-gal substrate after cell fixation, whereas
untransfected cells remained unstained. Transfection efficiency was
estimated by selecting 10 random fields on the microscope and counting
the percentage of blue cells versus unstained cells.
Plasmid Constructions--
Dominant negative JUN (dn-JUN) was
originally from Dr. M. J. Birrer (10) The constitutively active
form of JUN (v-JUN) was a gift from Dr. N. Hay (University of Chicago).
Both dn-JUN and v-JUN were tested; the expected results were obtained
when an AP-1 reporter plasmid 3x-TRE-CAT was used, and there was no
effect on pBL-CAT. Plasmid 3x-TRE-CAT contains three AP-1 sites to
regulate CAT gene expression. The control empty parental vector pBL-CAT has no AP-1 sites. Both plasmids were kindly provided by Drs. B. J. Aneskievich and E. Fuchs (21). The colCAT reporter plasmid p1.7/1.6
contains 1.7 kilobases of the 5'-UPS of the rat
I(I) collagen gene
and 1.6 kilobases of the first exon and part of the first intron linked
to the CAT reporter gene (22, 23). Plasmids p1.3/1.6 and p0.4/1.6
colCAT reporters were derivatives of plasmid p3.6/1.6 produced by
digestion with NheI/TthIII I and NheI/MfeI restriction endonucleases,
respectively. Plasmids p3.6/1.6 and pdel1.3-0.4/1.6 were as described
previously (2). pdel 1.4-0.4 was generated by PCR. The upstream primer
(
1506 to
1487) was
5'-CACCTAGCTAGCGGAATCTTGGATGGTTTGG-3'. Twelve additional nucleotides with an NheI restriction site were added to the
5'-end of the primer. The downstream primer (
1412 to
1429) was
5'-CCTCAATTCAGGCCATAGACGTCCTGTATC. Twelve additional
nucleotides with an MfeI restriction site were added 5' of
the primer. The generated plasmid pdel 1.4-0.4/1.6 was sequenced to
ensure that no changes occurred during the PCR.
PCR, DNA Sequencing, and Site-directed Mutagenesis--
PCR was
performed using Ultma DNA Polymerase (Perkin-Elmer). The sequencing
procedures followed the protocol of Sequenase Version 2.0 DNA
Sequencing Kit from Amersham Pharmacia Biotech. Plasmid p1.7(GC box
mut)/1.6 was created by the overlap extension method of site-directed
mutagenesis (24, 25). The site-directed mutants were sequenced to
confirm the mutations. The site (
1494 to
1468) was mutated from
5'-GGTTTGGAGGAGGCGGGACTCCTTGC-3' to
5'-GGTTTGGAGGAAATAAGACTCCTTGC-3'. This site contains the GC
box of interest (i.e.
1484 to
1475)
Nuclear Extraction--
Nuclear proteins were prepared as
follows. >95% confluent cells in cell culture flasks were washed
twice and harvested in cold PBS. Cells were collected by centrifugation
at 2000 rpm for 5 min at 4 °C. The cell pellet was resuspended in
5-10× volume of Solution A, which contained 10 mM Hepes,
pH 7.9, 1.5 mM MgCl2, and 10 mM
KCl, and incubated for 10 min at 4 °C. Cells were re-collected by
centrifugation for 5 min at 2000 rpm. The cell pellet was resuspended in 1.5-2 ml of Solution A with the following inhibitors: 0.5 mM dithiothreitol, 10 µg/µl leupeptin, 2 mM
phenylmethylsulfonyl fluoride, 10 mM NaF, 10 µg/ml
aprotonin, 1 mM NaVO3, and 60 mM
-glycerophosphate. Cells were gently stroked 30 times on ice in a
Dounce Type B homogenizer. Nuclei were collected by centrifugation at
2500 rpm for 10 min at 4 °C. The nuclei pellet was resuspended in
Solution A with the above inhibitors and subsequently centrifuged at
15,000 rpm for 20 min at 4 °C. The pellet was resuspended in Solution C, containing 20 mM Hepes, pH 7.9, 25% glycerol,
420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 µg/µl
leupeptin, 2 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 10 µg/ml aprotonin, 1 mM
NaVO3, and 60 mM
-glycerophosphate. The
nuclei suspension was incubated on an up-and-down rocker for 60-90 min
at 4 °C and then centrifuged at 15,000 rpm for 30 min. The clear
supernatant was aliquoted in microcentrifuge tubes and stored at
70 °C until use. Nuclear protein concentrations were quantified by
Bio-Rad reagent.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear protein
extracts (5-10 µg) were incubated in 10 µl of binding buffer with
4% glycerol, 1 mM MgCl2, 0.5 mM
dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH
7.5, and 2.5 µg of poly(dI-dC). In competition EMSAs, the indicated
amounts of double-stranded oligonucleotides were added as unlabeled
competitor. The mixture was incubated at room temperature for 10 min
before adding a radiolabeled probe. In some cases, 2 µl of anti-BTEB
or preimmune serum was added to the mixture and incubated at room
temperature for 20 min prior to adding 1 ng of a radiolabeled probe.
Nucleoprotein complexes were resolved by electrophoresis on 6%
nondenaturing polyacrylamide gels in 0.5x Tris borate/EDTA (TBE)
buffer. The gels were exposed to autoradiography film (DuPont)
overnight at
70 °C. Double-strand oligonucleotide probes were
radiolabeled by T4 polynucleotide kinase (New England
Biolabs) and [
-32P[dATP (DuPont). Polyclonal antibody
-BTEB was commercially generated by Research Genetics, Inc.,
according to the rat BTEB cDNA and its deduced amino acid peptide
sequence (26).
Oligonucleotide Probes--
The double-strand oligonucleotides
used in the EMSA were as follows. SP-1 consensus oligonucleotides
purchased from Promega: 5'-ATTCGATCGGGGCGGGGCGAGC-3'; GC
box oligonucleotides from
1491 to
1470 of the 5'-UPS of the rat
I(I) collagen gene: 5'-TTGGAGGAGGCGGGACTCCTTG-3'. Underlined sequences represent GC box binding domain. All of the oligonucleotide probes were synthesized by Life Technologies, Inc.
Southwestern Blot Analysis--
Nuclear extracts (100 µg/lane)
were obtained either from HSCs cultured in DMEM with 20% or 0.4%
serum for 48 h or directly from normal uncultured rat HSCs or rats
pretreated with carbon tetrachloride (CCl4) (0.5 ml + 0.5 ml of mineral oil given intraperitoneally) 16, 24, 48 or 72 h
prior to sacrifice. The extracted proteins and prestained protein
standards were resolved by 15% SDS-polyacrylamide gel electrophoresis
and transblotted to a nitrocellulose membrane filter. The filter was
washed for 30 min in Buffer A (25 mM NaCl; 25 mM Hepes-NaOH, pH 7.9; 5 mM MgCl2;
0.5 mM dithiothreitol, which was added fresh prior to use).
The filter was then blocked by incubation for 3 h at room
temperature in Buffer A with 5% nonfat milk. After a short wash with
Buffer A containing 0.25% nonfat milk, the filter was probed by
32P-labeled double-strand GC box oligonucleotide (from
1491 to
1470 of the 5'-UPS of the rat
I(I) collagen gene)
5'-TTGGAGGAGGCGGGACTCCTTG-3' in 10 ml of Buffer A with 5 µg/ml
poly(dI-dC) plus 0.25% nonfat milk on a rocker at 4 °C overnight.
The filter was briefly washed twice with Buffer A containing 0.25 M NaCl and then washed with regular Buffer A twice at room
temperature for 15 min each. The filter was wrapped and exposed to
autoradiography film (DuPont) overnight at
70 °C.
Statistics--
Differences between means were analyzed via
Student's t test using the Statworks program for the
Macintosh. Statistical significance required differences at the level
of p < .05.
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RESULTS |
JNK Activation by UV Irradiation Increases Endogenous
I(I)
Collagen Gene Expression--
The effects of activation of JNK on
I(I) collagen gene expression were studied in passaged HSCs treated
with UV irradiation (Fig. 1). HSCs were
grown to near confluence and then incubated for 48 h in
serum-depleted medium (0.4% serum). The cells were then exposed to
ultraviolet light for 0.2 or 0.4 min or to regular light for 0.4 min.
The cell extracts were prepared for Western blot analysis (Fig.
1A). It was found that the active forms of JNK 1 and JNK 2 were readily detected in HSCs exposed to UV light by anti-activated JNK
polyclonal antibody (Promega), which preferentially recognized the
dually phosphorylated active forms of JNK enzymes (Fig. 1A).
In contrast, faint bands of active forms of JNK were present in HSCs
exposed to regular light (Fig. 1A). UV irradiation did not
induce activation of MAPK (ERK1,2) in serum-starved HSCs probed with
the anti-activated MAPK polyclonal antibody (Promega) (data not shown).
To determine the effects of UV-induced activation of JNK on endogenous
I(I) collagen gene expression in HSCs,
I(I) collagen mRNA
from serum-starved HSCs treated with or without UV irradiation was
measured by RPA. (Fig. 1B). The radioactivity in each band
in the RPA was measured and quantitated by computer-aided densitometry
of the phosphorimage using IPLab Gel. As shown in Fig. 1C,
it demonstrated that JNK activation by UV irradiation caused an
approximate 2.9-fold increase in endogenous
I(I) collagen mRNA
steady state levels in HSCs.

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Fig. 1.
UV irradiation activated JNK and increased
endogenous I(I) collagen gene expression in HSCs. A,
serum-starved HSCs were exposed to UV light for 0.2 min or 0.4 min, or
to regular light for 0.4 min as control (ctr) before
incubation in medium with 0.4% serum for an additional 1 h.
Fifteen micrograms of total cell lysate proteins per lane were
separated in 10% SDS-polyacrylamide gel electrophoresis and analyzed
by Western blots with ANTI-ACTIVE JNK polyclonal antibody.
B, 10 µg/lane of total RNA from cells exposed to UV light
(UV) or to regular light (no UV) for 0.4 min were
analyzed by RPA. Lane P is the undigested control RNA
probes. Arrows at the right indicate protected
I(I) collagen and cyclophilin mRNA. Representative assays are
shown here. C, the radioactivity in each band in
B was quantitated by computer-aided densitometry of the
phosphor image using IPLab Gel. Cyclophilin was used as an internal
control to normalize the loading of RNA in each lane.
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Activation of JUN Stimulates colCAT Expression in Cultured
HSCs--
To understand the mechanisms of UV induction of
I(I)
collagen gene expression, a series of transfection experiments were performed in passaged HSCs (Fig. 2). HSCs
were transfected with an AP-1 reporter plasmid 3x-TRE-CAT or an empty
parental plasmid pBL-CAT (Fig. 2A). The Ap-1 reporter
plasmid 3x-TRE-CAT has three AP-1 binding sites to regulate CAT gene
expression, whereas the pBL-CAT is the control parental plasmid without
AP-1 binding sites. These two plasmids have been used previously to
study activation of AP-1 and transcription mediated by AP-1 (27). After
transfection, the cells were incubated in media containing 0.4% serum
for 36 h before exposure to UV light or regular light. The results
indicated that UV irradiation induced a >4-fold increase in the CAT
activity in cells transfected with 3x-TRE-CAT (Fig. 2A). In
contrast, UV exposure did not change the CAT activity in HSCs
transfected with pBL-CAT (Fig. 2A). These results suggested
that UV irradiation increased the ability of the AP-1 complex to
regulate gene transcription. To study the significance of the
activation of JUN in stimulating
I(I) collagen gene expression, HSCs
were co-transfected with colCAT plasmids and a v-JUN expression plasmid
(Fig. 2B) or a dn-JUN plasmid (Fig. 2C). In Fig.
2B, the results illustrated that v-JUN, but not empty
control plasmid pMNC, increased the CAT activity in serum-starved HSCs
co-transfected with the p1.7/1.6 colCAT reporter plasmid, which
contained 1700 base pairs of the 5'-UPS and 1600 base pairs of the
first exon and part of the first intron of the
I(I) collagen gene
linked to a CAT reporter gene. However, v-JUN did not increase the CAT
activity in HSCs co-transfected with the p0.4/1.6 colCAT plasmid (Fig.
2B). Fig. 2C showed that dn-JUN had an opposite
effect on p1.7/1.6 in HSCs maintained in medium with 20% serum. dn-JUN
significantly reduced the CAT activity in HSCs co-transfected with
p1.7/1.6 but not in HSCs co-transfected with p0.4/1.6. The lack of the
inhibitory effect in HSCs co-transfected with p0.4/1.6 suggests that
the necessary response element is missing and that the dn-JUN
inhibitory effect is not due to a nonspecific sequestration effect. It
should also be emphasized that v-JUN experiments were carried out in
0.4% serum conditions to eliminate serum derived sources of JUN
stimulation, whereas the dn-JUN experiments were done in the presence
of serum (20%) as a source of JUN stimulation. Taken together, these
data indicate that activation of JUN plays an important role in
increasing
I(I) collagen gene transcription in vitro and
that the response element for activated JUN is located between
1.7
and
0.4 kilobases of the 5'-UPS of the
I(I) collagen gene.

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Fig. 2.
Activation of JUN-stimulated colCAT
expression in HSCs. A, HSCs were transfected with AP-1
reporter plasmid 3x-TRE-CAT or control plasmid pBL-CAT. The cells were
incubated for 36 h in DMEM with 0.4% serum before exposure to UV
light or to regular light for 0.4 min. B, HSCs were
co-transfected with colCAT p1.7/1.6 or p0.4/1.6 and v-JUN or control
plasmid pMNC. The cells were incubated in DMEM with 0.4% serum for
36 h before harvest for CAT assay. C, HSCs were
co-transfected with colCAT p1.7/1.6 or p0.4/1.6 and dn-JUN or control
plasmid pMNC. The cells were cultured for an additional 36 h in
DMEM with 20% serum. The CAT activity was measured by CAT assay (see
under "Materials and Methods" for details). Co-transfected
-galactosidase was used to normalize the transfection efficiency
(n = 6).
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The Distal GC Box in the 5'-UPS Is Required for JUN
Stimulation--
To further analyze the role of JUN in regulating
I(I) collagen gene expression in HSCs, and to identify the promoter
region responding to activated JUN, a series of colCAT reporter
constructs were produced, some of which are shown in Fig.
3A, left. In order to identify
the DNA response element, HSCs were co-transfected with dn-JUN and a
series of colCAT plasmids with different 5'-UPS truncations of the
I(I) collagen gene, and the experiments were performed in DMEM with
20% serum (Fig. 3A, right). dn-JUN resulted in a 2.6-fold
reduction in the CAT activity in cells co-transfected with p1.7/1.6
(Fig. 3A and statistical analysis in Table
I). A significant loss of responsiveness
to the dn-JUN inhibitory effect was observed in cells co-transfected
with p1.3/1.6 or p0.4/1.6 (Fig. 3A and statistical analysis
in Table I). Additional experiments were therefore focused on the
general region between
1700 and
1300 base pairs. To prove that this
region contained the required response element(s), additional
constructs were produced, i.e. pdel 1.3-0.4/1.6 and pdel
1.4-0.4/1.6 (see Fig. 3A for diagram). When transfected
with the p0.4/1.6 reporter, there was no response to the co-transfected
dn-JUN. In the pdel reporters, the region containing putative
responsive element(s) is now adjacent to the unresponsive 0.4/1.6
region. When transfected with these pdel plasmids, the response to the
dn-JUN was regained (see Fig. 3A). A computer-aided search
revealed a GC box binding site in this region (
1475 to
1484).
Because the GC box might be the element responding to dn-JUN, several
key nucleotides in the GC box were mutated to evaluate their necessity
for the dominant negative JUN inhibitory effect. As shown in Fig.
3A, this mutagenized reporter p1.7 (GC box mut)/1.6 lost the
inhibitory response to dn-JUN. This result suggests that the distal GC
box plays a key role in the response to the dn-JUN inhibitory effect on
I(I) collagen gene transcription. In order to confirm that the GC
box was the response element mediating the regulation of
I(I)
collagen gene expression by activated JNK, HSCs were co-transfected
with the v-JUN and colCAT p1.7/1.6 or p1.7(GC box mut)/1.6 plasmid
(Fig. 3B). v-JUN had a stimulatory effect on the CAT
activity in cells co-transfected with wild-type colCAT p1.7/1.6. The
cells co-transfected with p1.7(GC box mut)/1.6 lost the stimulatory
effects of v-JUN on CAT expression. Further evidence of GC box function
was provided by HSCs transfected with colCAT p1.7/1.6 or p1.7(GC box
mut)/1.6 before exposure to UV irradiation (Fig. 3C). The
col CAT plasmid with a mutated GC box (p1.7 (GC mut)/1.6) lost its
response to stimulation by activated JNK. These results collectively
suggest that the distal GC box in the 5'-UPS of the
I(I) collagen
gene is the response element required for UV activated JNK to stimulate
I(I) collagen gene expression.

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Fig. 3.
The distal GC box was required for colCAT
expression induced by JUN activation. A, colCAT
reporter constructs are shown schematically on the left. The
black bars reflect differing lengths of the 5'-UPS and the
first exon and part of the first intron of the I(I) collagen gene.
The black arrow indicates CAT reporter gene. The mutagenized
region in the distal GC box is shown as a diagonally striped
oval between 1500 and 1400 in p1.7 (GC box mut)/1.6.
Transfections for localizing the response region in the 5'-UPS for the
dn-JUN inhibitory effect are shown on the right. These
experiments were performed in DMEM with 20% serum as sources of JUN
stimulation (see details under "Materials and Methods").
B, GC box was required for activated JUN to induce colCAT
expression. Experiments here were carried out in DMEM with 0.4% serum.
C, GC box was required for UV irradiation to stimulate
colCAT expression. Experiments were done in DMEM with 0.4% serum. Data
for each transfection are expressed as the mean ± S.D.
(n = 6). For comparison, the CAT activity from
co-transfected control pMNC was normalized to 1.0. Transfection
efficacy was normalized by measuring co-transfected -galactosidase
activity (see under "Materials and Methods" for details).
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Table I
Dominant negative JUN suppression of collagen gene expression
Data are normalized by analyzing co-transfected -galactosidase
activity expressed as -gal relative unit/mg of protein (see under
"Materials and Methods") and expressed as mean ± S.D.
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GC Box-binding Protein Is Altered During Stellate Cell
Activation--
The GC box sequence is one of the most common
regulatory DNA elements of eukaryotic genes. A GC box is usually bound
by a member of the Sp transcription factor family (e.g. Sp-1
transcription factor) or BTEB (26). To analyze the GC box-binding
protein in HSCs, nuclear extracts were obtained from culture-activated HSCs and from HSCs isolated from rats injected with CCl4
(72 h prior to sacrifice). Injection of CCl4 results in HSC
activation typified by HSC proliferation and ultimately enhanced
I(I) collagen gene expression (3-5). The extracts from cells
activated in vitro and in vivo both contained a
single GC box-binding protein, which was demonstrated in the gel shift
assay as a single intense band with the same mobility (Fig.
4, lanes 6 and 11, lower
arrow). In contrast, a faint band with the same mobility was
present in quiescent HSC extracts obtained directly from normal rats
(lane 10) and in serum-deprived HSCs in culture (lane
5). Competition with either GC box oligonucleotides or a DNA
fragment from
1400 to
1500 base pairs of the 5'-UPS, which
contained the GC box eliminated this binding (lanes 7 and
8). Although Sp-1 binding could be involved in the GC box
binding, this did not appear to be likely. As shown in Fig. 4, an Sp-1
consensus oligonucleotide resulted in a gel shift that differed
considerably from the gel shift using the GC box probe (see Fig. 4,
lanes 1-3 versus lanes 6-11). Its mobility was slower, and
the response to serum-containing media was less significant. Also,
competition with excess Sp-1 oligonucleotide had a minimal effect on
the GC box gel shift (Fig. 4, lane 9). The GC box gel shift
appeared to require 72 h following CCl4 injection for
maximal activity (see Fig. 5). Extracts
from earlier time points were intact as they had the ability to bind to
a consensus Sp-1 domain (data not shown). This gradual onset of the GC
box-binding protein was consistent with the gradual increase in
collagen gene expression in vivo (4). The process might
require the recruitment of other cells and cytokine release to
adequately stimulate the HSCs (28).

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Fig. 4.
GC box gel shift coincided with stellate cell
activation. Nuclear extracts were prepared either from cultured
HSCs in medium with 0.4% serum (lanes 1, 3, and
5) or 20% serum (lanes 2 and 6-9) or
directly from freshly isolated HSCs without any treatment (lane
10) or with CCl4-injection 72 h prior to
isolation (lane 11). The nuclear extracts were incubated
with 32P-labeled Sp-1 consensus oligonucleotides
(lanes 1-3) or 32P-labeled GC box
oligonucleotides (lanes 4-11) (see under "Materials and
Methods" for details). In lane 4, there was no protein
extract. Lane 4 was used to indicate the position of the
free probe. In some assays, competition was performed with excess
(100×) of cold GC box oligonucleotides (lane 7), a cold DNA
fragment from 1400 to 1500 base pairs (lane 8), or cold
Sp-1 consensus oligonucleotides (lane 9). The lower
arrow on the left indicates a GC box gel shift band,
and the upper arrow indicates a Sp-1 gel shift band. Intact
proteins in the extracts are demonstrated in lanes 1 and
2.
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Fig. 5.
GC box gel shift versus time
post-CCl4. HSC extracts were prepared at different
time points, 6-72 h post-CCl4 injection. The GC box
oligonucleotide probe (GC box in the 1.4 to 1.5 kilobase region)
was identical to the probe used in Fig. 4. The arrow on the
right of the gel indicates a single shifted band.
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BTEB Is the GC Box-binding Protein--
The results of the EMSA
(Fig. 4) suggested that the DNA-binding protein is unlikely to be an
Sp-1 transcription factor because the nucleoprotein complex of interest
moved much faster than the Sp-1 complex in the gel. Further studies
were performed to identify the GC box DNA-binding protein in the 5'-UPS
of the
I(I) collagen gene in HSCs. The Southwestern blot revealed a
single 32-kDa protein, which could form a nucleoprotein complex with a
GC box oligonucleotide (Fig. 6). The
32-kDa size is incorrect for Sp-1 (29). The band also became prominent
at 72 h following CCl4 injection (Fig. 6, lane
6), which was consistent with our previous observation (Fig. 5).
As expected from the gel shift experiments, serum stimulation of
cultured HSCs also resulted in a GC box-binding protein that had the
same molecular mass (Fig. 6, lane 2). Recent studies
involving other cell types unrelated to HSCs had identified and cloned
a GC box-binding protein with a similar molecular mass referred to as
BTEB (26, 30). In another EMSA (Fig. 7),
nuclear protein extract was pretreated with polyclonal anti-BTEB. The
pretreatment with anti-BTEB significantly diminished the gel shift
retarded band of interest (lane 3). In great contrast,
pretreatment with preimmune serum had no effect on the abundance of the
retarded band (lane 4). These gel shift assays suggest that
BTEB is the distal GC box DNA-binding protein.

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Fig. 6.
Southwestern blot. Nuclear extracts were
prepared from HSCs cultured in medium with 0.4% serum (lane
1) or 20% serum (lane 2) or from freshly isolated HSCs
at different time points (6-72 h) post-CCl4 injection
(lanes 3-6). Blotted extracts were probed with the
32P-labeled double-stranded GC box oligonucleotides as
described in Fig. 4. The arrow on the left
indicates a single 32-kDa protein.
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Fig. 7.
Anti-BTEB pretreatment diminished the GC box
gel shift retarded band. The nuclear extracts were obtained from
HSCs maintained in medium containing 0.4% fetal calf serum (lane
1) or 20% serum (lanes 2-4). The extracts either had
no pretreatment (lanes 1 and 2) or were
preincubated with antiserum to BTEB (lane 3)
( BTEB) or with preimmune serum (lane 4)
(preI) followed by incubation with the radiolabeled GC box
oligonucleotide probe (see under "Materials and Methods").
Lane 5 had no nuclear extract, indicating the position of
the free probe.
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DISCUSSION |
The sinusoidal HSCs represent the major effector cells during
hepatic fibrogenesis. During this process, the normal quiescent, vitamin A-storing HSCs transform into actively proliferating, collagen-producing cells. The HSCs excessive collagen matrix production and increased cellular proliferation lead to the collagenization and
disruption of the space of Disse and formation of the fibrous septae
seen in cirrhosis. Eukaryotic cells have developed specific signal
transduction pathways to respond to and integrate extracellular stimuli. A recent study of HSCs found that fibronectin and tumor necrosis factor
activated both JNK and ERK1,2 and increased AP-1
DNA binding ability and
I(I) collagen mRNA abundance (16). However, that study could not clearly indicate the effects of JNK or
ERK1,2 on
(I) collagen gene expression because fibronectin and tumor
necrosis factor
activated both pathways. Our previous studies found
that activation of ERK1,2 induced by serum was important for the
maximal expression of
I(I) collagen gene in passaged HSCs (2). The
function of activated JNK in regulating
I(I) collagen gene
expression was studied in the present report. It was found that UV
irradiation activated JNKs, but not ERK1,2 (Fig. 1A) and
that UV irradiation increased endogenous
I(I) collagen mRNA
abundance in passaged HSCs (Fig. 1B). Both activated ERK1,2 and activated JNK have the capacity to translocate to the nucleus and
phosphorylate transcription factors (31, 32). The current transfection
studies indicated that activated JNK induced by UV irradiation
increased the ability of AP-1 to induce gene transcription in HSCs
(Fig. 2A). Additional studies suggested that the active form
of JUN increased
I(I) collagen gene transcription (Fig. 2B) and, as expected, dominant negative JUN inhibited
I(I) collagen gene transcription in cultured HSCs (Fig.
2C). The response element mediating UV induction of
I(I)
collagen gene expression was located in a distal GC box in the 5'-UPS
of the collagen gene.
A previous study found that an AP-1 binding site in the first intron of
the
I(I) collagen gene played a critical role in the stimulation of
the
I(I) collagen gene by transforming growth factor (TGF
) (33).
Another recent report indicated that the first intron of the
I(I)
collagen gene had a tissue-specific and developmentally regulated
function in transcription of the gene (34). In the present study, HSCs
transfected with plasmid p1.7/1.6 or p1.7 (GC box mut)/1.6 showed
significant differences in the CAT activity in their responses to
dominant negative JUN (Fig. 3A), to the constitutively
active form of JUN (Fig. 3B), and to UV irradiation (Fig.
3C). All of these experiments utilized plasmids containing
the intact wild-type AP-1 binding site in the first intron. The only
difference between the two plasmids was the distal GC box in the
5'-UPS. Five nucleotides inside the GC box were mutated by
site-directed mutagenesis in the plasmid p1.7 (GC box mut)/1.6. The
current study does not support the contention that the AP-1 binding
site in the first intron is directly involved in the induction of
I(I) collagen gene transcription by exposure of HSCs to UV light.
Further studies demonstrated that the GC box in the 5'-UPS of the
I(I) collagen gene was bound by BTEB, a recently described GC box
DNA-binding protein. A GC box sequence could be bound by either Sp-1 or
BTEB (26). BTEB does not share sequence similarity to Sp-1, except for
a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with 72% sequence similarity to Sp-1. The study by Imataka et al. (24) suggested that BTEB exerted different effects on gene transcription with differing numbers and positions of the GC box
sequence in the promoter region. Further studies are needed to
understand the relationship between BTEB functions and flanking nucleotides of the GC box. It is likely that BTEB binding to the GC box
plays a role in regulating
I(I) collagen gene expression because 1)
the GC box plays a contributory role in stimulating
I(I) collagen
gene expression (see Fig. 3), 2) BTEB appears to be the major protein
binding to the GC box at periods of increased
I(I) collagen gene
expression (Fig. 4), and 3) the appearance of BTEB was coincident with
activation of HSCs in both in vitro and in vivo
settings as demonstrated by the results shown in Figs. 5 and 6.
Additional studies are needed, such as co-transfections of HSCs with
colCAT plasmids and BTEB overexpression construct and experiments
involving BTEB overexpression and the measurement of endogenous
collagen gene expression in stable transfectants in non-HSC models.
There is little known about BTEB gene expression and regulation, and
its relationship to fibrosis and liver injury remains unclear. One
speculation is that activation of JNK and subsequent activation of JUN
directly transduces signals to the promoter of the BTEB gene through
JUN-JUN dimerization and then BTEB gene expression is stimulated. BTEB
could then bind to the GC box in the 5'-UPS of the
I(I) collagen
gene and stimulate gene transcription. These speculations will require
further studies.