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INTRODUCTION |
The laminins are a family of adhesive glycoproteins
composed of high molecular weight disulfide-bonded heterotrimers
composed of 

chains. To date, five
, three
, and two
chains have been described, forming at least 12 trimeric laminin
isoforms (1). Although laminins are distinguished by both subunit
composition and tissue distribution, the overall domain structure is
well conserved (1). Laminin 1 (EHS laminin) is an 820-kDa heterotrimer consisting of one each of
1 (400-kDa),
1 (220-kDa), and
1
(200-kDa) chains, and laminin 2 (Merosin) is an 800-kDa heterotrimer
formed of
2 (380-kDa),
1 (220-kDa), and
1 (200-kDa) chains.
Both covalent and noncovalent interactions contribute to the basic
structure of most laminins, a cruciform with three short arms and one
long arm (2). The individual laminin subunits share a common structural motif composed of repeated epidermal growth factor-like domains interrupted by globular domains.
A number of biological activities have been attributed to laminin,
including cell attachment, differentiation, and migration, along with
interactions with other matrix components (3). Laminin has also been
implicated in the process of tumor invasion and metastasis (4).
Different isoforms of the laminin molecule may vary with respect to
tissue distribution and developmental expression. The
1 and the
1
chains are expressed in most tissues that produce basement membranes,
but their ratios vary considerably (5).
In the liver, increased deposition of laminins, the main noncollagenous
glycoproteins in all basement membranes, was demonstrated in rats with
chemically induced carcinoma (6) and within the lobule of livers from
patients with malignancies (7). In contrast, in normal adult livers,
laminins are located mainly in the portal tracts and are only sparsely
deposited in the space of Disse (8). In human liver, the laminin
isoforms have not been clearly identified so far, and only information
from rodents is available. Rat hepatic stellate cells
(HSC),1 the main source of
extracellular matrix components in both normal and fibrotic livers,
express
1,
1, and an
chain of 380 kDa that probably
corresponds to unprocessed form of the
2 chain (9). Normal adult
hepatocytes do not express laminin 1 in vivo, but synthesize
both
1 and
1 chain mRNAs after a few hours in culture (6, 7).
Several hepatoma cell lines of either human or rat origin express both
1 and
1 chains at high levels and a 380-kDa polypeptide that is
genetically different from the
1 chain but stained by polyclonal
anti-laminin antibodies (10). Although most studies have focused on
laminins polymerized into a basement membrane, much laminin is found
freely soluble and diffusible. Furthermore, although the polymerization
of laminin in vitro occurs by a self-assembly mechanism,
basement membranes are formed at discrete sites close to the cell
membrane (11).
The 5'-flanking region of the mouse laminin
1
(LAM
1) gene has been cloned and characterized
(12). The LAM
1 gene appears to contain two
transcription start sites (
169 and
234) and it does not contain a
TATA or CAAT box, however, it has several interesting features,
including the presence of ten GC boxes, which act as putative binding
sites for the redox-sensitive transcription factor Sp1 (two in tandem
and the rest monomeric), and a stretch of nine nearly identical repeats
of 11 nucleotides between
200 and
450 with the sequence
5'-CCC(G/T)CCC(A/T)CCT-3'. The consensus sequence for cAMP
responsiveness is also present in the LAM
1
promoter and is similarly found in the promoters of other
extracellular-matrix proteins (12). These motifs act as transcription
activators in several extracellular-matrix genes. The integrity of the
CTC-rich region is required to promote LAM
1
activity. Consequently, it could be hypothesized that there is
interplay between CTC and GC elements, GC boxes being predominant in
the activation of truncated CTC-less LAM
1
promoter. The region from
830 to
224 appears to contain a negative
regulatory element, which decreases the promoter activity of the
LAM
1 gene. Deletion of
94 to
61
nucleotides reduced the promoter activity by severalfold in HepG2
cells, and deletion of 20 bp from
41 to
21 completely abolished the
promoter activity in HepG2 cells (13). On the other hand, the
LAM
1 promoter had a relatively high level of
activity in NIH-3T3 cells, which synthesize little laminin. This result
suggests that the 830-bp promoter segment may lack a negative
regulatory element, which inhibits transcription in certain cells
(12).
Ethanol and several other low molecular weight agents induce cytochrome
P450 2E1 (CYP2E1), which is of special interest because it metabolizes
and activates hepatic toxins as well as carcinogens and fatty acids
(14). CYP2E1 is a loosely coupled enzyme that generates high amounts of
reactive oxygen species (ROS) such as superoxide radical and
H2O2 (15, 16). Oxidative stress plays an
important role in mechanisms by which ethanol damages the liver (17).
Our laboratory has carried out studies to evaluate whether hepatocytes
with high levels of CYP2E1 can interact with nonparenchymal liver cells
such as HSC via release of diffusable mediators (18, 19). In the
present study we evaluated whether CYP2E1-derived oxidative stress
could modulate the expression of basement-membrane proteins such as
laminin, which are elevated during liver fibrosis, and the role of
oxidative stress in this inductive up-regulation of laminin.
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MATERIALS AND METHODS |
Cell Culture--
The model used in most of the experiments
described below is based on the co-culture of HepG2 cell lines that
express (E47 cells) or do not express (C34 cells) the human CYP2E1 (20,
21) with primary HSC or with an immortalized rat stellate cell line (HSC-T6). Primary HSC were isolated from male Sprague-Dawley rats (600 ± 25 g) (Charles River Breeding Laboratories, MA) by
in situ liver perfusion with bacterial collagenase and
pronase, followed by density-gradient centrifugation with Nycodenz
according to published protocols (22). Results of Figs. 2-5 were with
the primary HSC whereas other results were carried out with the HSC-T6
cells. Cell viability (95%) was assessed by the trypan blue exclusion method. Purity of the HSC fraction (97%) was determined as described previously (23). To extend results with the HepG2 cells to intact primary hepatocytes, selected experiments (Fig. 4) were carried out
using hepatocytes isolated from saline control rats or from pyrazole-treated rats whose CYP2E1 levels are elevated about 3- to
4-fold (24).
Details on the co-culture model are shown in Fig.
1. Cells were co-incubated using
cell-culture inserts of 3-µm pore size to separate both cell
populations; this allows study of the effect of diffusable
CYP2E1-derived mediators from the hepatocyte on HSC functions, which
resembles the physiological situation in the liver. The HSC were plated
on the bottom and the HepG2 cells or the hepatocytes on the filter to
create a gradient of the released mediators. The ratio of HepG2 or
hepatocytes to HSC was 5:1, similar to that of
parenchymal:nonparenchymal cells in liver. After overnight incubation
of HSC alone in minimum essential medium (MEM) supplemented with 10%
fetal bovine serum and essential amino acids, the HSC medium was
discarded, the cells were washed 5 times in MEM, and the cell-culture
inserts containing the HepG2 cells or the primary hepatocytes were
transferred along with their overnight culture medium. At this time all
additions were made (t = 0 h).

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Fig. 1.
Scheme of the co-culture model. HSC were
seeded on the bottom plate at a density of 2.5 × 105
cells in 3 ml of culture medium. CYP2E1 activity was monitored by the
p-nitrophenol oxidation method, and 1.25 × 106 C34 or E47 cells (in some cases primary hepatocytes)
were plated on the insert in 3 ml of culture medium. After overnight
incubation (typically 16 h), the medium from the HSC was
discarded, the cells washed 5 times in MEM, and the inserts were
transferred together with the incubation medium from the C34, E47, or
primary hepatocytes onto the HSC. New medium was added to HSC plated
with empty inserts; these were considered as non-co-cultured controls.
Various additions were made at this time (t = 0 h), and samples of HSC were collected at selected time points.
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General Methodology--
Plasmid DNA preparation as well as
transfection procedures, Northern and Western blot analysis, nuclear
run-on, the activities of antioxidant enzymes and glutathione (GSH)
levels were carried out as previously described (23, 25, 26). Mouse
laminin
1,
1, and
1 cDNA probes used for Northern blot and
nuclear run-on assays were kindly provided by Dr. Yoshihiko Yamada
(National Institutes of Health). The glyceraldehyde-3-phosphate
dehydrogenase cDNA clone was purchased from the American Type
Culture Collection. The Sp1 expression vector was a gift from Dr.
Robert Tjian (University of California, Berkeley). Western blots were
carried out routinely under denaturing conditions on cell lysates or
with incubation media using anti-laminin 1 antibody (1/5000), which
recognizes all
1,
1, and
1 subunits (Sigma) or anti-laminin
2 chain antibody (1/2000), which recognizes only the N-terminal
moiety of ~300 kDa and not the C-terminal moiety of ~75-80 kDa of
the
2 chain (Santa Cruz Biotechnologies). The anti-Sp1 (1/2000),
anti-tubulin (1/5000), and anti-fibrinogen (1/5000) antibodies were
from Santa Cruz Biotechnologies. Goat anti-rabbit IgG conjugated to
horseradish peroxidase was used as secondary antibody (1:5000;
Chemicon). In most of the Western blots, the laminin
1 and
1
chains run very close and were difficult to separate during the
electrophoresis (11), therefore bands have been labeled as laminin
1
and
1, except for the Western blot in Fig. 2A in which
both bands were clearly separated because of a longer run. Fig. 3,
C and D show Western blots run in nondenaturing conditions.
Laminin Synthesis and Turnover--
To assay the synthesis of
laminin
1 and
1 subunits, the HSC-T6 cells or HSC-T6 co-cultured
with the C34 or E47 cells were plated separately in 10% fetal bovine
serum-MEM; after 12 h, the medium from the HSC was removed and the
inserts containing the C34 or E47 cells together with their culture
medium were transferred onto the plates containing the HSC; fresh
medium was also added to the HSC that were plated with an empty insert
as a reference group (not to be co-cultured with HepG2 cells). 22 h later, the media from the complete co-culture systems were replaced
with methionine-cysteine-free MEM plus 10% dialyzed fetal bovine
serum, the cells were incubated for 2 h, after which they were
pulse-labeled with 150 µCi of EasyTagTM
EXPRE35S35S Protein Labeling Mix (PerkinElmer
Life Sciences) for 0, 2, 4, 8, and 12 h to study the synthesis of
laminin
1 and
1. A set of samples was pulsed for 2 h in the
presence of 40 µM cycloheximide, an inhibitor of protein
synthesis, as a control for the analysis of laminin
1 and
1
synthesis. The cells were washed in 1× phosphate-buffered saline and
lysed at the indicated time points with 150 µl of 10 mM
Tris-HCl buffer, pH 7.4, 0.5% Triton X-100, 1 mM EDTA, 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, and 1 mM phenylmethylsulfonyl fluoride.
To assay the turnover of laminin
1 and
1, defined as the loss of
[35S]methionine-labeled intracellular laminin
1 and
1 when HSC-T6 cells were chased with cold methionine, plus secretion
of [35S]methionine-labeled laminin
1 and
1 into the
medium, the co-cultures were treated as above but pulse-labeled with
the EXPRE35S35S mix for 24 h. The cells
were then washed three times and chased with complete MEM supplemented
with 300 µg/ml of cold methionine. Cells were washed in 1×
phosphate-buffered saline and lysed at 0, 1, 2, 4, 8, and 12 h in
the same lysis buffer. In all cases, laminin
1 and
1 was
immunoprecipitated with anti-laminin 1 IgG-protein G-agarose as
follows: 40 µg of protein of cell lysates and 1 mg of protein from
the culture medium were first incubated with 10 µl of preimmune
rabbit serum for 15 min, followed by the addition of 50 µl of a 50%
(v/v) suspension of protein G-agarose. After centrifugation for 2 min
at 13,000 rpm, the supernatant was incubated with anti-laminin 1 IgG by
rocking overnight at 4 °C followed by addition of 50 µl of a
suspension of protein G-agarose. Samples were centrifuged for 1 min at
13,000 rpm, the pellets were washed three times with lysis buffer, once
with lysis buffer plus 2% SDS, and three times with 0.1 M
Tris-HCl buffer, pH 6.8. Laminins
1 and
1 were eluted by boiling
for 5 min in Laemmli's buffer, samples were centrifuged for 2 min at
13,000 rpm to remove the protein G-agarose, resolved on a 5% SDS-PAGE,
and dried. The intensity of the radioactive signal was quantified using
PhosphorImager (Molecular Dynamics) and the ImageQuant software.
To evaluate total protein synthesis by HSC, cells were treated as above
and incubated with the
EasyTagTMEXPRE35S35S labeling mix
for 0, 2, 4, 8, and 12 h, followed by addition of 30%
trichloroacetic acid to stop the reaction. The cells were washed in 1x
phosphate-buffered saline, and the trichloroacetic acid-precipitable
counts were determined in a scintillation counter after resuspension of
the pellets in scintillation liquid. To evaluate loss of
[35S]methionine labeled HSC total protein, the
co-cultures were pulse-labeled for 24 h as above, followed by
chasing for 0, 1, 2, 4, 8, and 12 h. The HSC were treated with
30% trichloroacetic acid and washed, and the trichloroacetic
acid-precipitated counts were determined as described above.
Quantitative comparison of the intensity of the signal scanned in the
PhosphorImager was performed using ImageQuant software.
Construction of the Laminin
1-CAT Expression
Vectors--
These constructs were kindly provided by Dr. Yoshihiko
Yamada (National Institutes of Health) and were generated as described in Ref. 12: a recombinant plasmid, pKH 130, containing the promoter region of the laminin
1 chain gene (
833 to
+106) fused to the structural part of the gene encoding CAT
as a readout for transcriptional activity was constructed as follows: a
6-kb HindIII fragment containing the first exon and a single
NcoI site located at +300 bp was subcloned into PUC19. This
plasmid, pKH102, was linearized with NcoI and then partially
digested with the exonuclease Bal31. HindIII
linkers were attached, and the plasmid was self-ligated. One of the
plasmids, pKH121, containing 106 bp of the 5'-untranslated region was
selected. The 1-kb BamHI-HindIII fragment of
pKH121 whose BamHI site was converted to NdeI by
filling the end with Klenow fragment and attaching NdeI
linker was inserted into the NdeI-HindIII site of
pSVOCAT (27). The
1400LAM
1-CAT construct
contains a 1.4-kb laminin
1 promoter segment
(+106 to
1300) cloned into the HindIII site of pSVOCAT. To
generate the
2500LAM
1-CAT construct, the SmaI segment from the intron 1 was cloned into the
NdeI site of pKH130 (12).
Nuclear Protein Extraction and Electrophoretic Mobility-shift
Assays--
Nuclear extracts were prepared according to the method of
Dignam et al. (28). HSC were washed twice with 1×
phosphate-buffered saline and 100 µl of buffer A (10 mM
HEPES pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml bestatin, 10 µg/ml antipain, 10 µg/ml aprotinin, and 0.1 mM sodium orthovanadate, 0.6% Igepal-SC560) were added.
Cells were lysed for 15 min, and nuclei were isolated after
centrifugation for 20 s at 13,000 rpm. Nuclei were washed in
buffer A without Igepal-SC566, spun down for 20 s, the supernatant was discarded, and nuclear proteins were extracted in 40 µl of buffer
C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol,
0.2 mM EDTA, 25% glycerol, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
bestatin, 10 µg/ml antipain, 10 µg/ml aprotinin, and 0.1 mM sodium orthovanadate) by vortexing for 20 min at
4 °C. Samples were centrifuged for 10 min at 13,000 rpm, and the
supernatants were immediately frozen until analyzed.
For electrophoretic mobility-shift assays, synthetic oligonucleotides
for Sp1, AP1, and NF
B (Promega, Madison, WI) were end-labeled with
[
-32P]ATP and T4 polynucleotide kinase (Promega,
Madison, WI). Binding reactions were carried out in a total volume of
10 µl with 5 µg or 0.5 µg (for AP1) of the nuclear protein
extract from HSC, 1 µl of 10× binding buffer containing 1 µg/µl
of poly(dI-dC), and 20,000 cpm of labeled oligonucleotides at room
temperature for 30 min. For competition studies, 1000-fold cold
oligonucleotides were added along with labeled oligonucleotides.
Antibodies raised against Sp1, AP1 (c-Jun/c-Fos), and NF
B (p50/p65)
(Santa Cruz Biotechnologies) were used for supershifting
oligonucleotides-nuclear protein complexes. Polyacrylamide gel
electrophoresis (6%) was performed at 150 volts for 2 h in 0.5×
TBE (45 mM Tris borate/1 mM EDTA, pH 8).
Southwestern Blot Analysis--
Southwestern (DNA-protein)
blotting was performed by the method of Singh et al. (29).
Briefly, 25 µg of nuclear protein extract from HSC were
electrophoresed on a SDS exponential 5-20% gradient polyacrylamide
gel. After electrophoresis, the gel was soaked in 25 mM
Tris, 190 mM glycine pH 8.3, and 20% methanol for 1 h. Nuclear proteins were electrotransferred onto 0.2-µm nitrocellulose membranes using the same buffer containing 0.1% SDS.
After overnight transfer at 4 °C, the membranes were blocked in
Blotto containing 10% nonfat dry milk in TNE buffer (50 mM Tris pH 7.5, 40 mM NaCl, 1 mM EDTA). DNA
binding was carried out for 3 h with TNE buffer containing 5 µg/ml poly(dI-dC) and 2 × 105 cpm/ml
[
-32P]dCTP multiprime-labeled 80-bp fragment (
230 to
150) derived from the
2500LAM
1-CAT
construct by PCR amplification. Membranes were washed three times for 5 min each with TNE at room temperature and exposed in the PhosphorImager screen.
Nomenclature--
The designation HSC refers to data
found in the HSC incubated with an empty insert;
HSC/C34 refers to results obtained in the HSC
after co-culture with the C34 cells, whereas
HSC/E47 refers to results obtained in the HSC
after co-culture with the E47 cells.
Statistics--
Results are expressed as mean ± S.E. Most
of the experiments were repeated at least three times except for the
nuclear run-on, which is from one experiment only. Statistical
evaluation was carried out using Student's t test.
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RESULTS |
Laminin
1,
1, and
2 Protein Levels Increase in Primary HSC
Co-cultured with E47 Cells--
Primary HSC were co-cultured with
either the C34 or E47 cells. HSC lysates and aliquots of the incubation
medium were collected at 1, 2, 3, 4, and 5 d and analyzed by
Western blot for the expression of different laminin chains. A
time-dependent increase in laminin
1 (220 kDa) and
1
(200 kDa) production was observed in both systems but was higher in the
E47 co-culture when compared with the C34 co-culture (Fig.
2A). At 3 d of co-culture
there was a 2- to 4-fold increase in the HSC content of
laminin
1 and
1 chains, as well as a 3-fold increase in secretion of laminin
1 and
1 to the medium of the E47 co-culture as
assessed by Western blot (Fig. 2, B and C).
-Tubulin levels, as a control for loading, were identical in both
co-cultures. This difference in the amount of laminin
1 and
1
subunits in the culture medium in the E47 cell co-culture did not come
from laminin
1 and
1 produced by the E47 cells because the medium
from C34 and E47 cells cultured alone did not show any significant
differences in laminin
1 and
1 content (Fig. 2D). High
molecular weight bands in the 400-kDa region where laminin
1 would
appear could not be detected in any of the blots in Fig. 2,
B-D.

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Fig. 2.
Laminin 1 and
1 protein levels increase in primary HSC
co-cultured with E47 cells. Primary HSC were co-cultured with
either C34 or E47 cells for 1, 2, 3, 4, and 5 d and cell lysates
collected and analyzed by Western blot for laminin 1 and 1
expression (A). A representative Western blot of HSC lysates
(B) and culture medium (C) collected at 3 d
is shown. Incubation medium from C34 and E47 cells cultured alone did
not show any differences in laminin 1 and 1 secreted
(D). Arbitrary units under the blots refer to the intensity
of the laminin 1 and 1/ -tubulin ratio or the laminin 1 and
1/fibrinogen ratio.
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When the same samples were blotted and incubated with anti-laminin
2
chain antibody, laminin
2 chain expression was observed, and higher
expression of laminin
2 was detected intracellularly at 3 d in
HSC co-cultured with E47 cells as compared with the
HSC/C34 co-culture (Fig.
3A) as well as in the culture
medium (Fig. 3B). When the same cell extracts were
electrophoresed under nondenaturing and nonreducing conditions, a
dimer/s of about 400 kDa was observed after 3 d of culture,
whereas a heterotrimer of about 800 kDa was observed after 5 d but
not 3 d (Fig. 3, C and D) of culture. There
was increased formation of the dimer/s at 3 d and the heterotrimer at 5 d in the HSC/E47 co-culture compared with the
HSC/C34 co-culture, in agreement with the increase in the
individual
1,
1, and
2 laminin chains. Of note is the
observation that the 800-kDa heterotrimer band was not detectable until
day 5 of co-incubation with the HepG2 cells, which suggests that
assembly of the
2 subunit (whose expression is also increased under
higher oxidative stress conditions) with the
1 and
1 subunits
appears to be delayed with respect to the increase in levels of
expression of the individual
2,
1, and
1 laminin chains or the
400-kDa dimer/s. The laminin heterotrimer is believed to play the major
role in laminin's action as a basement-membrane protein. Whether the
presence of extracellular nonassembled
2,
1,
1 chains has any
physiological role is not known.

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Fig. 3.
Expression of laminin
2 chain in the co-cultures. Western blot
showing the expression of laminin 2 chain in HSC co-cultured for
3 d with either C34 or E47 cells. A, intracellular
expression of laminin 2 chain. B, levels of the 2
chain secreted into the incubation medium. C and
D, Western blot of HSC lysates under nondenaturing
conditions. HSC lysates after 3 d (C) and 5 d
(D) from the co-cultures were electrophoresed under
nondenaturing conditions and incubated with anti-laminin 1 antibody.
The same blots were incubated for -tubulin as a control for loading.
Arbitrary units under the blots refer to the ratio of
laminin/ -tubulin. For D, arbitrary units refer to the
ratio of the 800-kDa band/ -tubulin.
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ROS Mediate the Induction of Laminin
1 and
1 in Primary
HSC Co-cultured with E47 Cells--
To determine whether
the increase by co-culturing HSC with E47 cells on laminin
1 and
1 expression was ROS-mediated, the co-cultures were treated
for 24 h with either 2000 units/ml catalase to decompose
H2O2 or with 50 µM vitamin E to
prevent lipid peroxidation, and laminin
1 and
1 protein content
was analyzed by Western blot. Fig.
4A shows that both catalase
and vitamin E prevented the increase in laminin
1 and
1 subunits
in HSC co-incubated with E47 cells; these antioxidants also
decreased laminin
1 and
1 in the HSC/C34 co-culture.
Catalase and vitamin E had no effect on the
-tubulin loading control
levels. Thus, laminin
1 and
1 levels appear to be sensitive to
induction by ROS. To validate that the increase in laminin
1 and
1 levels found with the HSC/E47 co-culture was indeed
because of CYP2E1, the co-cultures were incubated in the presence of
CYP2E1 inhibitors such as 5 mM diallylsulfide, 2 mM 4-methyl pyrazole, 0.1 mM sodium
diethyldithiocarbamate, and 10 µM phenylisothiocyanate.
The increase in laminin
1 and
1 production by the E47 co-culture
was significantly lowered by the CYP2E1 inhibitors (Fig.
4B). These inhibitors produced only minor changes in the C34
system (which lacks CYP2E1 expression), validating their specificity
for CYP2E1. Moreover, transfection with antisense CYP2E1 cDNA
blocked the increase of the E47 co-culture on laminin
1 and
1
levels in HSC without any effect in the HSC/C34 co-culture, whereas transfection of C34 or E47 cells with sense CYP2E1
further increased laminin
1 and
1 production in HSC about 2-fold
(Fig. 4B). Transfection with empty plasmid (pCI-neo) had no
effect. To confirm that these CYP2E1 inhibitors and transfection experiments modulated CYP2E1 levels and activity, CYP2E1 expression was
determined by Western blot analysis and its activity measured by the
catalytic oxidation of p-nitrophenol to
p-nitrocatechol. The CYP2E1 inhibitors lowered CYP2E1
activity by 70-80%, transfection with antisense CYP2E1 decreased
CYP2E1 activity by 80%, whereas transfection with sense CYP2E1
elevated CYP2E1 activity 3-fold (data not shown). These results
indicate that the increase in laminin
1 and
1 content in the
HSC/E47 co-culture is mediated via CYP2E1 acting through a
ROS-dependent mechanism.

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Fig. 4.
Antioxidants and CYP2E1 inhibitors prevent
the induction of laminin 1 and
1 in the E47 co-culture. Primary HSC were
incubated with C34 or E47 cells in the presence or absence of 2000 units of catalase or 50 µM vitamin E for 24 h, HSC
were lysed, and laminin 1 and 1 expression was analyzed by
Western blot (A). Both systems were treated with the CYP2E1
inhibitors 5 mM diallylsulfide (DAS), 2 mM 4-methyl pyrazole (4MP), 0.1 mM
sodium diethyldithiocarbamate (DETC), or 10 µM
phenylisothiocyanate (PITC). HSC were also transfected
with plasmids containing the cDNA for CYP2E1 in the sense and
antisense orientation. Laminin 1 and 1 expression in HSC lysates
was evaluated by Western blot analysis. Arbitrary units under the blots
refer to the intensity of the laminin 1 and 1/ -tubulin
ratio.
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Hepatocytes from Pyrazole-treated Rats Increase Laminin
1 and
1 Protein in HSC--
To validate the results obtained
with the HepG2 cell lines with primary hepatocytes, freshly isolated
primary HSC were co-incubated with primary hepatocytes from either
control or pyrazole-treated rats. Pyrazole induces CYP2E1 protein
expression in hepatocytes about 3- to 4-fold over the levels present in
saline control hepatocytes and stabilizes the protein against
degradation (24). Laminin
1 and
1 protein levels increased 3-fold
and more than 6-fold in HSC cultured with saline- and
pyrazole-hepatocytes, respectively, when compared with HSC cultured
alone (Fig. 5, basal conditions, lanes
marked as "
"). This increase by both co-cultures was prevented by
added catalase and vitamin E, indicating the involvement of ROS. In
addition, the increase in laminin
1 and
1 proteins by both
co-cultures was decreased by CYP2E1 inhibitors: 5 mM
diallylsulfide, 0.1 mM sodium diethyldithiocarbamate, and 10 µM phenyl isothiocyanate (Fig. 5). These results
suggest that CYP2E1-derived ROS contribute to the increase in laminin
1 and
1 protein content of HSC in the saline-hepatocyte/HSC
co-culture, and to the further increase produced by the
pyrazole-hepatocytes/HSC co-culture.

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Fig. 5.
Hepatocytes from pyrazole-treated rats
increase laminins 1 and
1 proteins in HSC. Freshly isolated HSC were
placed in culture with no further treatment or co-cultured with primary
hepatocytes from either saline- or pyrazole-treated rats. Pyrazole
induces CYP2E1 protein expression in hepatocytes and stabilizes the
protein against degradation. Some cells were treated with antioxidants
such as 2000 units/ml catalase or 50 µM vitamin E, or
with the CYP2E1 inhibitors 5 mM diallylsulfide
(DAS), 0.1 mM sodium diethyldithiocarbamate
(DETC), or 10 µM phenyl isothiocyanate
(PITC) at the time of plating. HSC lysates were collected
and analyzed for laminin 1 and 1 content by Western blot.
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CYP2E1-derived Diffusible Mediators Increase the Synthesis of
Laminin
1 and
1 Proteins by HSC but Do Not Affect the
Turnover of Newly Synthesized Laminin
1 and
1--
The results
described above indicate that co-culture of HSC with E47
cells increases laminin
1 and
1 protein levels. This effect could
involve transcriptional activation of the LAM
1
and LAM
1 genes with elevated mRNA
synthesis and/or mRNA stability, increased translational
efficiency, or decreased turnover of newly synthesized laminin
1 and
1 protein, with subsequent accumulation in the HSC. To
address these possibilities, direct analysis of laminin
1 and
1
protein synthesis, turnover, and mRNA levels by the co-cultures was
performed. For the subsequent experiments, because of the need for
large amounts of HSC and because transfection experiments with reporter
constructs were to be used, a HSC-T6 cell line with a higher efficiency
for transfection was used rather than primary HSC.
Laminin
1 and
1 protein synthesis was assessed by labeling with
[35S]methionine for varying times, up to 12 h,
followed by immunoprecipitation with anti-laminin 1 IgG as described
under "Material and Methods." There was a strong increase in the
incorporation of [35S]methionine into laminin
1 and
1 in the HSC/E47 co-culture compared with HSC cultured
alone or in the presence of the C34 cells (Fig.
6, A and B).
Cycloheximide blocked laminin
1 and
1 synthesis in all systems,
validating that the increase in the laminins
1 and
1
[35S]methionine signal was caused by a protein
synthesis-dependent reaction. Total protein synthesis by
HSC was not altered by co-culturing with E47 cells (Fig.
6C). These results suggest that an increase in laminin
1
and
1 synthesis is associated with the induction of both proteins in
the presence of CYP2E1-mediated oxidative stress. Although synthesis of
the laminin
2 chain was not determined in these experiments, Western blot analysis confirmed that laminin
2 chain levels were elevated after 12 h of culture with the HSC/E47 co-culture
(Fig. 6D), analogous to the increase found with primary HSC co-cultured with E47 cells (Fig. 3A).

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Fig. 6.
Synthesis of intracellular laminin
1 and 1 in HSC in the
co-cultures. The synthesis of laminin 1 and 1 was determined
after 0, 2, 4, 8, and 12 h of incubation with
[35S]methionine and immunoprecipitation of 40 µg of
intracellular proteins as described under "Material and Methods"
and in Ref. 19. (A). A set of experiments in which cells
were incubated with 40 µM cycloheximide was included at
2 h. B and C, synthesis of intracellular
laminin 1 and 1 and total protein, as determined by
PhosphorImager analysis of fluorographs of immunoprecipitated
laminin 1 and 1 or by counting cell pellets after trichloroacetic
acid precipitation. D, Western blot for intracellular
laminin 2 subunit after 12 h of incubating HSC alone or with
C34 or E47 cells.
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To study the apparent turnover of laminin
1 and
1 chains, the
HSC were first labeled with [35S]methionine
for 24 h and, after washing, chased with excess unlabeled methionine for up to 12 h. Laminins
1 and
1 proteins were
immunoprecipitated at time points of 0, 1, 2, 4, 8, and 12 h, and
the remaining intracellular incorporated label quantified by
SDS-polyacrylamide gel electrophoresis and fluorography. The decrease
in total intracellularly labeled protein was determined by
trichloroacetic acid precipitation, washing, and counting the
solubilized HSC pellet for [35S]methionine in
trichloroacetic acid-precipitable protein. The apparent turnover of the
newly synthesized laminin
1 and
1 subunits and total proteins was
calculated from the semilogarithmic plot of counts incorporated per
minute versus time. Prior to the initiation of the chase (0 h), there was an increase in [35S]methionine-labeled
laminin
1 and
1 in the HSC/E47 co-culture (as
described above) (Fig. 7, A
and B). Pulse-chase experiments revealed that the turnover
of newly synthesized laminin
1 and
1 (reflected as time for 50%
loss of intracellular [35S]methionine counts) was
5.6 h for stellate cells cultured alone, 5.7 h for stellate
cells co-incubated with C34 cells, and 6.0 h for stellate cells
co-cultured with E47 cells (Fig. 7, A and B).

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Fig. 7.
Turnover of intracellular laminin
1 and 1 proteins made by
the HSC in the co-cultures. A, turnover of laminin 1
and 1 was studied in HSC cultured alone or with C34 or E47 cells
after pulsing with [35S]methionine for 24 h,
followed by chasing with complete MEM supplemented with cold methionine
for 0, 1, 2, 4, 8, and 12 h. Immunoprecipitation of 40 µg of HSC
protein with anti-laminin 1 antibody, washing, and fluorography was
carried out as described under "Materials and Methods" and in Ref.
19. Laminin 1 and 1 levels were determined from PhosphorImager
analysis of fluorographs shown in A, whereas total protein
turnover was determined from the decline in trichloroacetic
acid-precipitable counts. B and C, turnover of
intracellular laminin 1 and 1 and total protein,
respectively.
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Loss of intracellular radioactivity incorporated into laminin from the
HSC labeled with methionine could reflect turnover of the
labeled laminin
1 and
1 proteins because of intracellular
degradation or secretion into the medium or both. To evaluate the
latter, samples of media from the pulse-chase experiment were collected
at the same time points, and immunoprecipitation of laminins
1 and
1 chains was carried out as described above (Fig.
8). Very low rates of secretion of the
newly synthesized laminin were observed over the 12-h chase under these
conditions. In fact, 25 times more protein had to be immunoprecipitated
to observe the bands shown in Fig. 8A than for the results
of Fig. 7A. The loss of intracellular cpm as a function of
time cannot be accounted for by the small secretion of newly
synthesized laminin
1 and
1 during the 12-h chase as the increase
in total medium cpm was just a few percent of the decline in total
cellular cpm. The amount of laminin secreted to the culture medium was
minimal in both co-culture systems. Yurchenco et al. (11)
expressed the
,
, and
chains subunits of laminin 1 in all
combinations in a near-null background, and showed that in the absence
of its normal partners, the
chain is secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the
and
chains, expressed separately or together, remain
intracellular with formation of 
or 
, but not 
,
disulfide-linked dimers. Secretion of the
and
chains required
simultaneous expression of all three chains and their assembly into


heterotrimers. They concluded that the
chain can be
delivered to the extracellular environment as a single subunit, whereas
the
and
chains cannot, and that the
chain drives the
secretion of the trimeric molecule. Such an
chain-dependent mechanism could allow for the regulation of
laminin export into a nascent basement membrane, and might serve an
important role in controlling basement-membrane formation. As mentioned
above, the laminin
1 chain was not observed in the HSC, whereas the
2 chain was detected both in cell lysates and in the culture medium,
suggesting that the laminin
2 chain was driving secretion of the
1 and
1 subunits into the medium. Importantly, the experiments
described above suggest that turnover of laminin
1 and
1 proteins
was similar in both co-culture systems and not likely to explain the
increase in laminin
1 and
1 proteins by the HSC/E47
co-culture. Turnover of total HSC proteins were also
similar (about 3.6 to 3.9 h) for HSC cultured alone, or
co-incubated with C34 or E47 cells (Fig. 6C). There were no
differences in the secretion of total proteins in both co-culture
systems (Fig. 7C).

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Fig. 8.
Secretion of laminin
1 and 1 during
the pulse-chase in the co-cultures. A, secretion of
laminin 1 and 1 into the medium was determined at the same
time-points indicated for the pulse-chase (Fig. 7), immunoprecipitating
1 mg of total protein with the anti-laminin 1 antibody. Laminin 1
and 1 levels were determined from PhosphorImager analysis of
fluorographs shown in A, whereas total protein secretion was
determined from the trichloroacetic acid-precipitable counts.
B and C, secretion of laminin 1 and 1 and
total protein, respectively. The cpm refers to total accumulation in
the medium at the indicated time point as the medium was not changed
during the 12 h incubation.
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Induction of Laminin
1 and
1 Proteins in the E47 Co-culture
Involves Transcriptional Regulation--
To determine why laminin
1
and
1 synthesis was elevated, total RNA was isolated from
HSC-T6 cultured alone or with C34 or E47 cells and was
analyzed by Northern blot for laminin
1,
1, and
1 chains
mRNAs. Laminin
1 mRNA was not detected in any of the cell
models. The HSC/C34 co-culture resulted in an increase in
laminin
1, but not
1 mRNA levels over mRNA levels in the
HSC cultured alone (Fig. 9A).
The HSC/E47 co-culture produced an increase in laminin
1
mRNA as well as further induction of laminin
1 mRNA.
Overall, there was a 2- to 3-fold increase in
1 and
1 laminin
mRNA levels in HSC co-cultured with E47 cells compared
with the HSC/C34 co-culture (Fig. 9A). Nuclear
in vitro transcription assays were performed to study
whether the CYP2E1-mediated effect on laminin
1 and
1 mRNA
levels in HSC is regulated at the transcriptional level. As
shown in Fig. 9B, enhanced laminins
1 and
1 expression
occurs through a transcriptional mechanism with both co-culture
systems, however, the newly transcribed laminin
1 and
1 mRNAs
were increased in HSC co-incubated with E47 cells when
compared with HSC co-incubated with C34 cells. It is not
understood why laminin
1 transcription appears
to be up-regulated in the HSC/C34 co-culture compared with
HSC alone, whereas the mRNA levels are similar.

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Fig. 9.
Induction of laminin
1 and 1 in the E47
co-culture involves transcriptional regulation. A,
total RNA was isolated from HSC cultured alone or with C34 and E47
cells and analyzed by Northern blot for laminin 1, 1, and 1.
Laminin 1 mRNA was not detected. Numbers below the blot refer to
the ratio of arbitrary densitometric units of laminin 1 or
1/GAPDH. B, nuclear in vitro
transcription assays for laminin 1 and 1 were carried out with
newly transcribed mRNA from HSC cultured alone or with C34 or E47
cells. The signals of newly transcribed GAPDH and S14 mRNAs were
used as housekeeping genes. There was a slight increase in both the
GAPDH and S14 signals by the C34 or E47 cell co-cultures perhaps
reflecting a general effect by factors released from hepatocytes on
stellate cell transcription. However, the increase was the same for
both co-cultures, and results were normalized to account for the slight
increase in the loading controls. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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Identification of the Sequences of the LAM
1 Promoter in
HSC Required for CYP2E1-mediated
Responsiveness--
Transient-transfection experiments in
HSC-T6 with chimeric constructs harboring progressive 5' deletions of the LAM
1 promoter linked to the
CAT reporter gene were performed to identify the regions of
the LAM
1 promoter required for
CYP2E1-dependent activation. HSC cells were
transfected with the constructs shown in Fig.
10. The percentage of acetylation of
chloramphenicol in the HSC co-cultured with E47 cells and
transfected with the
330LAM
1-CAT and the
1400LAM
1-CAT was significantly higher (about
46-fold) than in HSC cultured alone or with C34 cells (Fig. 10,
A and B). These data are consistent with previous
findings (Fig. 9, A and B) that
CYP2E1-dependent LAM
1 activation
is exerted, at least in part, at the transcriptional level.
Interestingly, the activity of the
330LAM
1-CAT and
1400LAM
1-CAT constructs were very similar (about 45% acetylation of chloramphenicol). On the other hand, the
activity of the
580LAM
1-CAT (and
2500LAM
1-CAT) constructs was significantly
lower (4% of acetylation of chloramphenicol) and very close to the
basal levels of promoter activity by the shorter constructs
(
200LAM
1-CAT and
250LAM
1-CAT). These data suggest that the
230 to
480 region (and perhaps the
1400 to
2500 region) of the
LAM
1 gene may contain a silencer-like element which reduces promoter activity. Reporter activity was similar for HSC
incubated alone compared with the HSC/C34 co-culture for
the various constructs, which is in agreement with the similar laminin
1 mRNA levels found in the HSC compared with the
HSC/C34 co-culture (Fig. 9A). In general, the
pattern of promoter expression was similar for all three systems (HSC
alone, HSC/C34, and HSC/E47 co-culture),
i.e. all three systems showed positive response to the
330LAM
1-CAT and
1400LAM
1-CAT constructs, and negative
responsiveness to the
580LAM
1-CAT and
2500LAM
1-CAT constructs. However, the E47 co-culture clearly showed the most robust responses to the
330LAM
1-CAT and
1400LAM
1-CAT constructs, likely a reflection
of the presence of redox-sensitive sites in these regions. Thus, the
promoter responses are not unique for CYP2E1 effects but are enhanced
by CYP2E1-derived diffusable factors.

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Fig. 10.
Identification of the minimal sequences of
the LAM 1 promoter in HSC required for
CYP2E1-mediated responsiveness. Transient co-transfection
experiments of HSC with chimeric constructs harboring progressive 5'
deletions of the LAM 1 promoter linked to the
CAT reporter gene and with the null-RL luciferase vector
were performed in HSC cultured alone or with C34 or E47 cells.
A, representative blot for the chloramphenicol
acetyltransferase reaction and thin-layer chromatography. B,
schematic representation of the chimeric constructs together with the
percentage of acetylation of chloramphenicol corrected for protein
concentration and by transfection efficiency. ***, p < 0.001, compared with basal expression.   , p < 0.001, compared with HSC cultured alone or with C34 cells.
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CYP2E1-derived Oxidative Stress Transactivates the LAM
1 Promoter
in HSC Co-incubated with E47 Cells through a
Sp1-dependent Mechanism--
The
LAM
1 promoter possesses several putative
binding sites for redox-sensitive transcription factors including Sp1,
AP-1, and NF
B (30). To determine whether any of these transcription factors could, in response to the increase in ROS produced by the E47
co-culture, transactivate the LAM
1 promoter,
electrophoretic mobility-shift assays were carried out with nuclear
extracts from HSC incubated alone or cultured with either C34 or E47
cells (Fig. 11A and
inset). HSC co-cultured with E47 cells showed
increased binding of Sp1 to an oligonucleotide containing its putative
binding site GGGCGG when compared with HSC cultured alone or with C34
cells. There were no changes in the binding activity of NF
B and AP-1
(The inset shows an electrophoretic mobility-shift assay
loading only 0.5 µg of protein from the same samples). Competition
studies with a 1000-fold excess of cold Sp1 oligonucleotide blunted the
binding of the radiolabeled Sp1 oligonucleotide. The complex of Sp1
protein-DNA was supershifted with an anti-Sp1 antibody, demonstrating
specificity of the complex (Fig. 11A, last lane).
Southwestern analysis carried out with a double-stranded
oligonucleotide obtained by PCR amplification of the
230 to
150
region of the LAM
1 promoter (where strong reporter activity was noted, Fig. 10) and nuclear proteins from HSC cultured alone, with C34 cells, or with E47 cells,
showed a single band of about 100 kDa, which corresponds to the
molecular mass of Sp1 (95-106 kDa) for all samples (Fig.
11B). The binding activity was comparable between the HSC
and the HSC/C34 co-culture but was 2-fold higher in
HSC incubated with E47 cells. The increase in binding
activity for the E47 co-culture was prevented by the CYP2E1 inhibitor
diallylsulfide and by the free-radical-scavenging agent tempol (Fig.
11B). To verify that the band was indeed Sp1, a Southwestern
analysis was carried out in which the membrane was incubated with an
excess of anti-Sp1 antibody before hybridization with the
double-stranded DNA oligonucleotide. No signal was obtained (data not
shown), indicating that the detected band was Sp1, and suggesting that
the protein mediating the CYP2E1 effects on the
LAM
1 promoter may be Sp1. To eliminate the
possibility that increased binding and transactivation of the
LAM
1 promoter could be caused by increased
levels of Sp1 in the HSC co-cultured with E47 cells, a
Western blot analysis of total Sp1 protein was carried out with nuclear
protein extracts. Results in Fig. 11C show that no
differences in Sp1 protein content were observed in HSC
cultured alone or with C34 or E47 cells. Finally, co-transfection of
HSC with an Sp1 expression vector plus the
330LAM
1-CAT reporter construct, which
contains seven putative binding sites for Sp1 and whose binding
activity was induced about 8-fold in the HSC/E47 co-culture
compared with the C34 system (Fig. 10B), were carried out
(Fig. 11D). Although the co-transfection with the Sp1
expression vector enhanced reporter activity in all three systems (HSC
alone, HSC/C34, and HSC/E47), HSC transfected
with the Sp1 expression vector and co-cultured with E47 cells showed
higher CAT activity for the
330LAM
1-CAT reporter construct than did HSC cultured alone or with C34 cells (about
5-fold) (Fig. 11D). A Western blot analysis was also carried out to validate that Sp1 levels were elevated after transfection and
levels were comparable in the three HSC systems (Fig. 11D, lower panel).

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Fig. 11.
CYP2E1-derived oxidative stress
transactivates the LAM 1 promoter in HSC
co-incubated with E47 cells through a Sp1-dependent
mechanism. A, electrophoretic mobility-shift assays
were carried out with nuclear extracts (5 µg of protein) from HSC
cultured alone or with C34 or E47 cells using radiolabeled
oligonucleotides containing the consensus sequence for either Sp1,
AP-1, or NF B as described under "Material and Methods." The
Sp-1/DNA complex was competed with a 1000-fold of cold probe and
supershifted with an anti-Sp1 antibody. The inset shows an
electrophoretic mobility-shift assay for AP-1 carried out with 0.5 µg
of nuclear extract. B, Southwestern analysis was carried out
using an [ -32P]dCTP double-stranded oligonucleotide
obtained by PCR amplification of the 230 to 150 region of the
LAM 1 promoter, and nuclear protein extracts
from HSC cultured alone or with C34 cells, or with E47 cells that were
incubated in the absence or presence of 5 mM diallylsulfide
(DAS), a CYP2E1 inhibitor, or 10 µM tempol, a
spin trap agent. C, Western blot analysis showing no
differences in the expression of Sp1 protein in HSC in the three cell
culture systems. D, CAT assay with cell extracts from HSC
co-transfected with a Sp1-expression vector plus the
330LAM 1-CAT deletion construct. A Western
blot analysis of the Sp1 levels after transfection is shown at the
bottom of the figure.
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DISCUSSION |
Basement membranes are cell-associated heteropolymers that are
essential for tissue development and maintenance. The functions of
these extracellular matrices are both architectural and informational, with basement membranes acting as substrata, filters, and solid-phase agonists (31-33). In the liver, laminin is present during maturation of the organ and accumulates in the adult during the capillarization process that occurs in alcoholic cirrhosis and hepatocarcinogenesis (8,
34). Laminin
1 mRNA is abundant in HSC, the major site of matrix
formation, and it is also expressed at high levels in transformed
hepatoma cell lines and during chemically induced hepatocarcinogenesis
in the rat (6, 35), but it is not present in normal hepatocytes (8).
The presence of laminin
1 chain is a prerequisite for
basement-membrane formation, with its absence causing an early
embryonic lethality (36). Besides self-assembling, laminins also
interact with other laminin isoforms as well as with nonlaminin
extracellular matrix molecules such as perlecan, nidogen, and collagen
to form a polymeric matrix; such associations appear necessary for
proper basement membrane assembly (1).
Previous work in our laboratory has been aimed at characterizing the
intercellular communication between the hepatocyte and the
stellate cell to better understand the mechanisms by which oxidative
stress and other mediator molecules perpetuate the fibrogenic response
in stellate cells (18, 19). A co-culture model containing HSC with a
HepG2 cell line that overexpresses cytochrome P450 2E1 (E47 cells) or a
control HepG2 cell line (C34 cells) was developed. The interest in
CYP2E1 was due to its activation of numerous hepatotoxins, generation
of oxidative stress, and possible role in contributing to
alcohol-induced liver injury (14, 17, 37-42). This co-culture system,
with constant generation of ROS, revealed increased translation of
collagen mRNA by a ROS-dependent mechanism. Because of
its importance as an abundant extracellular matrix component whose levels are elevated during liver injury, we extended our co-culture studies to the possible regulation of laminin production under oxidative stress conditions generated by CYP2E1.
Kleinman et al. (43) have shown that normal adult liver
contains both laminin
1 and
1 chains but lacks the
1 chain.
Initial results shown in Figs. 2 and 3 revealed a
time-dependent increase in both intra- and extracellular
laminin
1,
1, and
2 proteins (after 3 days of culture) which
was enhanced in HSC co-cultured with E47 cells
compared with the HSC/C34 co-culture. The laminin
1
chain could not be detected, confirming its lack in normal liver (43).
To show interactions between the
1,
1, and
2 laminin chains,
Western blots of HSC lysates collected after 3 or 5 d of
co-culture were carried out under nondenaturing conditions. The laminin
1 antibody used recognizes the
1,
1, and
1 chains of laminin.
Two bands of about 400 and 800 kDa were found after 5 d of
culture, whereas the 400-kDa band was found after 3 d of culture.
The 400-kDa band may likely be the dimers
1/
1,
1/
1, and/or
1/
1, because the molecular mass of the
1 chain is about
220 kDa and that of the
1 chain is about 200 kDa. The 800-kDa band
may likely be the heterotrimer
2
1
1 because the molecular mass
of the
2 chain under native conditions would be of about 380 kDa
plus 220 kDa of the
1 and 200 kDa of the
1. Although other
chains (
3,
4, and
5) have been described, none of them have
been detected in the liver except for the
2 chain (9). Of note is
the fact that although there seems to be a time-dependent
increase in laminins
2,
1, and
1 with higher levels of
expression in the E47 co-culture, the potential assembly of the three
subunits to the 800-kDa heterotrimer is delayed, relative to the
increase in levels of the individual chains, or to the assembly into
dimers of
1 and
1 (400 kDa).
The amount of H2O2 and lipid peroxidation end
products in HSC or in the medium was previously shown to be
increased by the E47 cell co-culture (18, 19). The antioxidant defense
in HSC did not change with any of the culture conditions
(data not shown). Evidence for oxidative stress involvement in the
increase in laminin
1 and
1 proteins by the E47 co-culture is
based on the prevention of these effects by addition of antioxidants
such as catalase or vitamin E to the incubation medium and by CYP2E1
inhibitors such as diallylsulfide, 4-methyl pyrazole, sodium
diethyldithiocarbamate, and phenylisothiocyanate. Transfection of E47
cells with an antisense CYP2E1 construct lowered laminin
1 and
1
protein expression to basal levels, whereas transfection with a sense
CYP2E1 plasmid into E47 or C34 cells further increased laminin
1 and
1 expression. The relevance of these findings was further extended
to co-cultures of HSC with primary hepatocytes from pyrazole-treated rats, with high CYP2E1 content, when compared with
co-cultures with hepatocytes from saline-treated rats.
Experiments were carried out to determine the mechanism(s) responsible
for the increase in laminin
1 and
1 levels by the E47 co-culture.
The synthesis of laminin
1 and
1 chains was similar between HSC
cultured alone or with C34 cells, but an increase in synthesis was
found in HSC cultured with E47 cells. This effect was
blocked by cycloheximide. The turnover of laminin
1 and
1
proteins was similar in HSC cultured alone or with C34 or
E47 cells, and the export of laminin
1 and
1 chains to the
culture medium was very low during the 12-h chase; thus, changes in
laminin
1 and
1 degradation and/or secretion do not account for
the increase in laminin
1 and
1 proteins. These results indicate
that increased synthesis of laminin protein is one mechanism of
regulation for the induction of laminin
1 and
1 protein by the
E47 co-culture. We next analyzed whether elevated mRNA levels could
account for the increase in synthesis of laminin protein found in the
E47 system. Northern blot analysis revealed elevated laminin
1 and
1 mRNAs in the E47 compared with the C34 co-culture. Nuclear
run-on experiments documented increased synthesis of both mRNAs.
Thus, enhanced laminin
1 and
1 expression in the E47 system
results from transcriptional activation of the
LAM
1 and LAM
1 genes.
There is specificity in the ability of CYP2E1-derived mediators to
interact with the HSC and stimulate the synthesis of
certain proteins, e.g. collagen (19) and laminin, whereas
total protein synthesis is not altered, nor is the synthesis of other
HSC proteins such as catalase, tissue inhibitor of metalloproteinase 1, or metalloproteinase 13 altered (19).
Consistent with these results, transient transfection of
HSC co-cultured with E47 cells with chimeric constructs
driven by different sequences of the LAM
1
promoter indicated the presence of two redox-sensitive enhancer
elements located in the
230 to
150 and
1300 to
480 regions that
were not stimulated in HSC cultured alone or with C34 cells. Little data are available on the molecular mechanisms involved in the transcriptional regulation of basement-membrane genes in liver fibrosis. Laminin is highly expressed in hepatic fibrosis (44). The
5'-untranslated region of LAM
1 contains
a stem-loop structure spanning from +76 to +106. Deletion of 47 bp
within the 5'-untranslated region (+59 to +106) of the
LAM
1 completely blocked promoter activity in
astrocytes, confirming that this downstream region could be one of the
major points of transcriptional regulation (30). The chimeric
constructs used for transient-transfection studies in the
HSC systems contained the 5'-untranslated region of the
first exon, which is GC-rich and has a stem loop structure; whether
these could be redox-sensitive and operate coordinately with other
factors released by the E47 system is not known.
The regulation of the expression of laminin
1 mRNA in hepatoma
cells involves several regions within the 2-kb promoter, and transfection of LAM
1 promoter fragments in
these cells indicated that regulatory elements are located between
594 and
94 bp (35). The
230 to
150 region of the promoter
contains several monomeric Sp1-binding sites and a cAMP-responsive
element. The LAM
1 gene promoter contains
multiple cognate sites for Sp1 binding which have the ability to
recruit other transcription factors to initiate transcription from
TATA-less promoters (45). TATA-less promoters typically have multiple
transcription-initiation sites that are located very close or within
the regions that contain Sp1-binding sites. The multiple Sp1-binding
sites in these classes of genes suggest that they could be
redox-sensitive promoters. Overexpression of Sp1 in normal hepatocytes
increases endogenous LAM
1 gene expression and
co-transfected LAM
1 promoter (46).
High-binding activity was observed in Sp1-transfected nuclear extracts.
Sp1 and laminin
1 mRNA are both highly expressed in human
hepatocarcinomas, particularly at the invasive front (47).
In view of the above, we evaluated Sp1-binding activity in the
different HSC systems. Electrophoretic mobility-shift
assays showed increased Sp1-binding activity in nuclear extracts from
HSC co-incubated with E47 cells compared with that of HSC
cultured alone or with C34 cells. The DNA-protein complex was shifted
by an anti-Sp1 antibody and competed by a 1000-fold excess of cold
oligonucleotide containing the Sp1-binding site. Two other well known
redox-sensitive transcription factors, AP-1 and NF
B, showed the same
binding activity in all three systems. The AP-1 site located at
650
bp is not involved in LAM
1 transcription in
hepatoma cells, whereas several sequences in the
480-bp to
175-bp
region have been identified and may bind specific regulatory factors,
including Sp1 and immediate early gene products coded by the
erg-family (48). The results described above suggest that
the increased promoter activity in transient-transfection studies with
the
230 to
150 reporter construct could be mediated by increased
binding of the redox-sensitive transcription factor Sp1 to this region.
To verify this, we performed Southwestern analysis to determine the
binding capacity of the
230 to
150 region of the promoter to
nuclear proteins from HSC cultured alone or with C34 or E47
cells. This region of the promoter binds to a protein of about 100 kDa,
and the binding is increased 2- to 3-fold in HSC
co-cultured with E47 cells. A role for a CYP2E1-mediated effect and for
ROS was validated by addition of diallylsulfide, a CYP2E1 inhibitor,
and of 4-hydroxy-tempo (tempol), a wide-spectrum free radical
scavenger, both of which were able to prevent the enhanced Sp1 binding
of the E47 co-culture. Sp1 is a dimer of molecular masses 95 and 106 kDa. To determine whether this binding protein was Sp1, the same
samples were analyzed by Southwestern blot incubating the membrane with
anti-Sp1 antibody before hybridization with the
230 to
150
double-stranded oligonucleotide; no binding was detected after
incubation with the antibody. To ensure that the increase in Sp1
binding observed was not a result of increased Sp1 synthesis, a Western
blot analysis was carried out with nuclear proteins from HSC cultured
alone or with C34 or E47 cells, but no differences were observed among
the three systems. These results suggest that the increased
LAM
1 promoter activity found with the
HSC/E47 co-culture transfected with the
230 to
150
construct could be due to increased Sp1 binding. Furthermore, in
experiments in which a Sp1 expression vector was co-transfected along
with the
330LAM
1-CAT reporter construct, a
5-fold increase in CAT activity was detected in the HSC/E47
co-culture compared with HSC cultured alone or with C34 cells.
The LAM
1 gene is transcriptionally
up-regulated by interleukin-1
due to an increased binding of NF
B
to a
B consensus sequence on the LAM
1
promoter (49). Both interleukin-1
and TGF-
transiently increase
laminin
1 mRNA due to enhanced binding of nuclear proteins on
the GC-rich bcn-1 motif in the promoter. The cooperative induction of
the LAM
1 promoter and the endogenous
LAM
1 gene by TFE3 and Smad3 is augmented by
the TGF-
signaling pathway (50). In our reporter assays, there was
decreased activity in all systems (HSC alone, HSC/C34, and
especially HSC/E47 co-culture) with the
2500LAM
1-CAT construct, suggesting the presence of a silencer-like element between the
1300 and
2400 region. Not much is known about the sequence of the
LAM
1 promoter upstream of
1000 bp. We have
not analyzed possible redox-sensitive elements further upstream that
could be responsible for the transactivation of the
1400 to
480
region of the promoter. Whether this could be mediated directly by ROS
or involve other factors such as cytokines, most of which are
redox-sensitive molecules, still remains to be elucidated.
In summary, these results suggest that CYP2E1, present in the
hepatocyte, can release diffusible mediators, most likely stable ROS
such as H2O2 and lipid peroxidation
metabolites, which can up-regulate the LAM
1
gene, with a subsequent increase in synthesis of laminin
1 and
1
proteins. Up-regulation of the LAM
1 gene is
due, in part, to enhanced Sp1 binding to the
230- to
150-bp promoter region, which contains several redox active binding sites. The
enhanced Sp1 binding appears to be due to redox activation of Sp1 via
CYP2E1-derived diffusible ROS. Such up-regulation of important
matrix-synthesizing genes may play a role in liver injury produced by
hepatotoxins activated by CYP2E1, e.g. CCl4,
benzene, acetaminophen, nitrosamines, and perhaps in mechanisms of
alcohol-induced liver injury.