Department of Medicine, Veterans Affairs Medical Center, and Center for Molecular Genetics, University of California, San Diego, California 92161
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ABSTRACT |
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We analyzed the
role of oxidative stress on liver collagen gene expression in vivo.
Long- and short-term supplementation with the lipophilic antioxidant
D--tocopherol (40 IU/day for
8 wk or 450 IU for 48 h) to normal C57BL/6 mice selectively decreased liver collagen mRNA by ~70 and ~60%, respectively. In transgenic mice, the
0.44 kb of the promoter and the first intron of the human collagen
1(I) gene were sufficient to confer responsiveness to D-
-tocopherol. Inhibition
of collagen
1(I) transactivation in primary cultures of quiescent
stellate cells from these transgenic animals by
D-
-tocopherol required only
0.44 kb of the 5' regulatory region. This regulation
resembled that of the intact animal following D-
-tocopherol treatment and
indicates that D-
-tocopherol
may act directly on stellate cells. Transfection of stellate cells with
collagen-LUC chimeric genes allowed
localization of an "antioxidant"-responsive element to the
0.22 kb of the 5' region excluding the first intron. These
findings suggest that oxidative stress, independently of confounding
variables such as tissue necrosis, inflammation, cell activation, or
cell proliferation, modulates in vivo collagen gene expression.
liver fibrosis; antioxidants; transcription; stellate cells
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INTRODUCTION |
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ALTHOUGH OVERPRODUCTION OF collagen type I by hepatic
stellate cells (lipocytes) is a critical step in the development of liver cirrhosis (15, 19, 20, 35), the regulation of collagen 1(I)
gene expression remains unclear (12). From several lines of
investigation, we have obtained evidence indicating that
aldehyde-protein adducts, including products of lipid peroxidation,
modulate collagen gene expression (6, 13, 25) and may be a link between
injury and fibrosis (3, 24, 28).
Coculture experiments of hepatocytes and stellate cells treated with
carbon tetrachloride (a hepatocyte, but not stellate cell, toxin)
indicate that hepatocytes could exert a paracrine stimulation of both
lipid peroxidation and collagen gene expression on stellate cells (3).
In support of a role of reactive aldehydes on collagen gene expression,
both acetaldehyde (6) and malondialdehyde (13) increase collagen
1(I) gene transcription, and this effect can be blocked by
scavengers of reducing equivalents (13), which are required for the
formation of aldehyde-protein adducts. Acetaldehyde, a product of
ethanol oxidation, can also induce lipid peroxidation (43). In
agreement with these findings, stellate cell collagen gene expression
is stimulated by acetaldehyde (38) and by 4-hydroxynonenal (40),
another product of lipid peroxidation.
Whether oxidative stress plays a role in the modulation of collagen
gene expression in vivo is unknown. The assessment of this important
issue is complicated by the confounding variables of tissue necrosis
and inflammation associated with tissue injury. These confounding
variables could be avoided by analysis of constitutive collagen gene
expression. Because lipid peroxidation also occurs in normal tissues in
vivo (2, 10, 21, 25, 32), we investigated whether products of lipid
peroxidation might modulate basal liver collagen gene expression. In
this study, we show that
D--tocopherol supplementation
results in a decrease of collagen
1(I) gene expression in the
liver of normal mice and in cultured hepatic stellate cells.
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METHODS |
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Animal studies.
Three-week-old female C57BL/6 mice received either a
D--tocopherol-supplemented
diet (8 IU/g of foodstuff) or a control (Purina Chow) diet for 8 wk.
Transgenic mice received intraperitoneal injections of either
D-
-tocopherol (100 mg, 150 IU) or equal volumes of mineral oil (200 µl) at 0, 24, and 48 h and
were killed 6 h after the last injection.
Expression of chimeric collagen 1(I)
human growth hormone reporter genes in the liver.
Transgenic mouse lines expressing human growth hormone (hGH) transgenes
were as described by Bornstein and co-workers (34, 44). We used a
transgenic line containing portions of the human
1(I) collagen
gene,
2300COL
I (bases
2300 to +1607, with deletion of
most of the first intron) and
440COL (bases
440 to +1607, which include the first intron and first exon). The
440COL line has been previously described (26, 44) and contains 2 copies of the
transgene, whereas the
2300COL
I line contains 10-12
copies (26). The cis-regulatory region
of the collagen
1(I) gene responsive to
D-
-tocopherol was
characterized in transgenic mice expressing hGH under the direction of
2300 or
440 bp of the human collagen
1(I) without or
with the first intron, respectively (44). At least three animals were
used for each group in all experiments.
Cell cultures.
Primary hepatic stellate cells from transgenic mice were isolated
essentially as described previously (26). Briefly, after the livers
were excised and washed in Hanks' balanced salt solution (HBSS), they
were minced and incubated at 37°C for 30 min with constant shaking
with 0.5% Pronase (Boehringer Mannheim), 0.05% collagenase B
(Boehringer Mannheim), and 10 µg/ml DNase (United States Biochemical)
in HBSS without Ca2+. This digest
was filtered through gauze and pelleted at 450 g for 10 min and subsequently rinsed
four times with HBSS containing 10 µg/ml DNase. Primary hepatic
stellate cells from rats were isolated as described previously (3, 33).
Stellate cells were purified by a single-step density Nycodenz gradient
(Accurate Chemical & Scientific, Westbury, NY), as described previously (3, 26, 33). Cells were cultured on EHS (Matrigel) and used after 6 days of culture. In some experiments, stellate cells growing on an EHS matrix were activated with
FeSO4 (50 µM)-ascorbate (200 µM), which induces oxidative stress, as we previously
described (33). Cells were cultured under an atmosphere of 5%
CO2 and 95% air in DMEM
containing 10% FCS. Stellate cells were identified by their typical
autofluorescence at 328 nm (excitation wavelength), staining of lipid
droplets by oil red, and immunohistochemistry with a monoclonal
antibody against desmin (3). In addition, the activation of stellate
cells was determined by the expression of -smooth muscle actin,
judged by immunofluorescence using specific antibodies against
-smooth muscle actin (Sigma) (33).
Determination of collagen 1(I) mRNA.
RNA was isolated from liver and quantitated by a sensitive RNase
protection assay, as described previously (7-9, 16, 26), using the
riboprobes for 18S RNA (Ambion), exon 5 of the hGH gene, and mouse
collagen
1(I) (26, 27, 44). Collagen
1(I) mRNA and hGH mRNA
values were measured simultaneously with the internal standard 18S RNA
as described by us previously (7-9, 16, 26, 27).
Statistical analysis. All the results are expressed as means ± SE. Student's t-test was used to evaluate the differences of the means between groups, accepting P < 0.05 as significant.
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RESULTS |
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We first analyzed the expression of the collagen 1(I) gene in the
liver of C57BL/6 mice that received a diet supplemented with the
lipophilic antioxidant
D-
-tocopherol (8 IU/g of
foodstuff, ~40 IU/day; n = 13) (8).
After 8 wk, the weight of animals receiving a control diet (Purina Chow
without the D-
-tocopherol supplementation; n = 7) was similar to
that of the D-
-tocopherol group (20 ± 1 vs. 21 ± 1 g; not significant). The effects of
D-
-tocopherol supplementation
on liver collagen
1(I) were analyzed by an RNase protection assay,
after purification of poly(A)+ RNA
from total liver RNA (5). The steady-state pool of collagen mRNA in the
liver was consistently and markedly inhibited (~70%) by dietary
supplementation with
D-
-tocopherol (Fig.
1, A and B).
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Because D--tocopherol
inhibits collagen
1(I) gene transcription in cultured human
fibroblasts within 72 h (25), we analyzed whether short-term
supplementation with
D-
-tocopherol would also effectively inhibit liver collagen gene expression.
D-
-Tocopherol (150 IU ip) was
administered at 0, 24, and 48 h to C57BL/6 mice. The endogenous
collagen
1(I) steady-state pool in the liver was decreased by
~60% in animals treated with
D-
-tocopherol compared with
control animals (receiving equal volumes of mineral oil vehicle) (Fig.
1C). Confounding variables present
during stellate cell activation in vivo and in culture (e.g., cytokine
production, cell proliferation) preclude a definitive characterization
of the "oxidative stress"-responsive region (3, 26, 33).
Therefore, to characterize the oxidative stress-responsive
cis-regulatory region within the
collagen
1(I) gene, we performed experiments with normal
transgenic mice expressing the hGH under the direction of regulatory
regions of the human collagen
1(I) gene (Fig.
2A) (44). Both the hGH transgene mRNA and the endogenous collagen
1(I)
mRNA were detected by a sensitive and specific RNase protection assay
(26). The expression of the
2300COL
I-hGH transgene and the
440COL-hGH transgene was markedly decreased in the liver, in
parallel to endogenous collagen (data not shown) after treatment with
D-
-tocopherol (150 IU ip at
0, 24, and 48 h) (Fig. 2B). In
normal liver, a 5' regulatory region that included only the
0.44-kb region was sufficient to direct the expression of the hGH. In the same animals,
D-
-tocopherol also inhibited
collagen
1(I) gene expression in tendon, a tissue with high
collagen production, by ~60% (K. S. Lee, unpublished
observations), suggesting that the bioavailability of
D-
-tocopherol and its
therapeutic efficacy, after only a 48-h treatment, are probably
adequate for modulating collagen gene expression in many
tissues.
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Because the effects of
D--tocopherol on liver
collagen gene expression could be exerted directly or indirectly on
stellate cells, we analyzed the modulation of collagen gene expression by D-
-tocopherol in freshly
isolated hepatic stellate cells. Stellate cells were obtained from
2300COL
I-hGH and
440COL-hGH transgenic animals as
discussed in METHODS. The stellate
cell populations were >95% pure, as determined by autofluorescence at 328 nm, conferred by retinoids. Contaminant cells were hepatocytes and other sinusoidal cells (3). To avoid the confounding variables of
cell activation and proliferation, which are associated with enhanced
oxidative stress (33), stellate cells were cultured on an EHS matrix.
Under this culture condition, stellate cells displayed for at least 6 days a quiescent phenotype similar to that of stellate cells in normal
liver, as described previously (26, 33). The expression of the
endogenous collagen
1(I) mRNA was inhibited by the
antioxidants
D-
-tocopherol and butylated hydroxytoluene (Fig.
3A).
Also, expression of hGH in stellate cells bearing the
2300COL
I-hGH (data not shown) or the
440COL-hGH was
markedly decreased from basal values by treatment with 50 µM
D-
-tocopherol during a 6-day
culture period (Fig. 3B). Thus transactivation of collagen
1(I) gene expression in primary
cultures of quiescent stellate cells required only
0.44 kb of
the 5' regulatory region and resembled the regulation of these
cells in the intact animal following
D-
-tocopherol treatment.
Complementary information about the role of lipid peroxidation on
collagen gene expression was obtained by treating control quiescent
stellate cells, cultured on EHS matrix, with ascorbic acid (200 µM)-FeSO4 (50 µM), a free radical-generating system (13), for 6 days. This treatment increased ~10-fold the mRNA steady-state pool of collagen
1(I) (data not shown). In addition, we have previously shown that
Fe2+-ascorbate induces hepatic
stellate cell activation (33). Taken together, these studies indicate
that basal oxidative stress modulates hepatic stellate cell collagen
1(I) gene expression in vivo and in culture and that the
0.44-kb region of the collagen
1(I) gene contains the
antioxidant-responsive element(s).
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The promoter of the collagen 1(I) gene is highly conserved among
different species, including humans, mice, and rats (14, 34, 42).
Therefore, additional characterization of the
cis-regulatory region within the
collagen 5' flanking sequences was obtained by transfecting a
LUC chimeric reporter gene driven by a
220-bp segment of the mouse collagen
1(I) gene into
activated (cultured in 20% serum) or quiescent (cultured in 0.5%
serum) primary rat stellate cells. Using a transfection-enhancing
reagent and lipofectamine, we achieved a high-efficiency transfection
for stellate cells, as discussed previously (27, 29). To obtain optimal
reporter expression, cells were harvested 48 h after transfection.
Expression of the LUC reporter
containing
220 bp of the 5' flanking region of the mouse
1(I) collagen gene (without the first intron) was inhibited by
D-
-tocopherol irrespective of
whether the cells were activated (20% serum) or quiescent (0.5%
serum) (Fig. 3C). The transfection
efficiency, as determined by a pLUC vector, was comparable in control
and D-
-tocopherol-treated
cells (12-16 U/µg DNA), indicating that
D-
-tocopherol did not
decrease collagen-chimeric reporter gene expression spuriously by
inhibiting the transfection of this gene.
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DISCUSSION |
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We have previously suggested that lipid peroxidation plays a critical
role in tissue fibrogenesis by stimulating collagen gene transcription
(3, 13, 24, 25). Moreover, we have shown that activation of cultured
hepatic stellate cells by transforming growth factor- and collagen
type I is mediated by oxidative stress (33). Here, we present evidence
strongly supporting the hypothesis that antioxidants, in the absence of
necrosis or inflammation, inhibit collagen
1(I) gene expression in
the liver of normal animals.
D-
-Tocopherol also inhibited
collagen
1(I) gene expression in cultured stellate cells,
independently of the confounding variables of activation stimuli.
The effects of D--tocopherol
were observed after an 8-wk supplementation of ~40 IU/day or after
only a 48-h treatment of 450 IU. These results suggest the feasibility
of controlling collagen gene expression in the liver during chronic or
acute induction of fibrogenesis. Furthermore, after a 48-h treatment,
D-
-tocopherol also inhibited
collagen gene expression in tendon, a tissue with high collagen
production. Altogether, these studies suggest that the bioavailability
of D-
-tocopherol is probably
adequate for many tissues and that
D-
-tocopherol inhibits
collagen gene expression in tissues with low and high rates of collagen
production. These findings could be of great relevance not only for the
treatment of excessive fibrogenesis in the liver but also in other
tissues such as lung, kidney, skin, pericardium, pleura, and peritoneum.
The precise molecular mechanisms by which oxidative stress regulates
collagen 1(I) expression in stellate cells are unknown. However,
oxidative stress plays an essential role, through the induction of
c-myb and nuclear factor-
B, on
stellate cell activation, including induction of
-smooth muscle gene
expression (33). Whether similar mechanisms also affect collagen
1(I) expression in stellate cells remains to be determined.
Reactive aldehydes are formed as a result of the oxidative breakdown of
polyunsaturated fatty acids, and these aldehydes may form covalent
links with various amino acid residues of proteins (4, 43). The
functions of various proteins are altered by the formation of
aldehyde-protein covalent bonds in vitro (4).
In this study, we also show that induction of free radicals with
ascorbic acid-FeSO4 increases
collagen gene expression in cultured quiescent stellate cells.
Malondialdehyde- and 4-hydroxynonenal-protein adducts have
been demonstrated on the induction of ascorbic
acid-FeSO4 in cultured
fibroblasts, and their formation is inhibited with D--tocopherol (13).
Presumably, by inhibiting lipid peroxidation and thus the formation of
reactive aldehydes such as malondialdehyde and 4-hydroxynonenal,
D-
-tocopherol inhibits adduct
formation. Presently, no other biologically important function has been
ascribed to D-
-tocopherol
distinct from its role as an antioxidant. In this context,
malondialdehyde- and 4-hydroxynonenal-protein adducts have been found
in animals with iron overload (24, 28) and CCl4-induced hepatotoxicity (3,
26) as well as in patients with genetic hemochromatosis (30) and
chronic viral hepatitis (31).
It is also important to note that aldehydes may form adducts with
virtually all cellular elements, including DNA (4, 43). Because
endogenous malondialdehyde-deoxyguanosine adducts have been detected in
apparently normal human livers (11), it is conceivable that aldehydes
may stimulate collagen gene expression by a direct interaction with DNA
cis-acting regulatory elements. Although expression of the collagen 1(I) gene in the liver during hepatocellular injury and lipid peroxidation induced by
CCl4 requires only the presence of
an upstream
0.44-kb region (26), it was uncertain whether this
transcriptional activation is mediated by oxidative stress or other
confounding factors. To characterize the regulatory region of the
collagen
1(I) gene responsive to oxidative stress, we analyzed the
expression of reporter chimeric genes in transgenic mice following
treatment with D-
-tocopherol. Here, we report that
D-
-tocopherol inhibits the
expression of the
440COL-hGH as well as of the
2300COL
I-hGH transgene in the absence of liver necrosis or
inflammation, strongly suggesting that the antioxidant effect is
exerted on the
0.44-kb flanking region of the collagen
1(I) gene.
Because in the intact animal
D--tocopherol could modulate
liver collagen
1(I) gene expression indirectly through paracrine mechanisms, we tested whether the same
cis-regulatory region of the collagen
1(I) gene contains the "oxidant"-responsive element(s) in
primary stellate cell cultures. In primary cultures of quiescent stellate cells (to avoid the confounding variables of cell activation and cell proliferation) bearing the
440COL-hGH transgene,
D-
-tocopherol inhibited the
expression of the hGH reporter to a degree similar to that observed in
the
440COL-hGH transgenic mice. These studies indicate that
D-
-tocopherol can directly
modulate stellate cell collagen expression through the
440-bp
segment of the 5' flanking region and/or the first
intron. Further characterization of the sequences responsive to
D-
-tocopherol was obtained by
transfection into stellate cells of a chimeric
LUC reporter gene. A regulatory region
of the collagen
1(I) gene containing the
220/+110-bp segment in the absence of the first intron was sufficient for the
inhibition of LUC reporter expression
by D-
-tocopherol in transfected primary stellate cells.
It was not known whether the mechanisms that modulate constitutive collagen expression in cultured cells also occur in vivo. Cells are usually exposed to a higher oxygen tension in vitro (1) than in vivo (22), and this could induce high levels of basal lipid peroxidation, which in turn may lead to a high constitutive collagen gene expression. However, under the conventional incubation conditions (95% air, 5% CO2), the level of lipid peroxidation found in cultured fibroblasts or myocardial cells (280 ± 30 pmol/mg protein) (17, 25) is comparable with that in normal skin (2, 32) and other tissues in vivo (10, 21, 25, 39). These findings suggest that the degree of lipid peroxidation is comparable in normal cultured cells and normal tissues in vivo. More importantly, basal oxidative stress appears to modulate constitutive collagen gene expression of quiescent hepatic stellate cells both in culture and in the normal liver.
Another important issue is whether the constitutive collagen gene
expression is spuriously high in cultured stellate cells or other
cells. Several studies seem to indicate that collagen gene expression
is quantitatively and qualitatively similar in vitro (6, 13, 18, 23,
41) and in vivo (45). Although absolute rates of collagen production
are difficult to measure, the data available suggest that the values
are equivalent (1-3 µmol proline · mg
protein1 · h
1)
in normal cultured fibroblasts (18, 23) and in normal skin in vivo (36,
37). Also, the steady-state level of collagen
1(I) mRNA by
solution hybridization is in the same range in stellate cells activated
in culture or in vivo (26).
We herein demonstrate that antioxidants, independently of other
confounding variables such as tissue necrosis, inflammation, cell
activation, or cell proliferation, modulate hepatic collagen gene
expression. Whether oxidative stress plays a role in the modulation of
constitutive liver collagen gene expression in humans is unknown. Given
the findings presented here, and due to the low toxicity of
D--tocopherol and other
antioxidants, the modulation of collagen gene expression by
antioxidants could now be assessed in normal individuals and eventually
in patients with active fibrogenesis in the liver or in other tissues.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. P. Bornstein (University of Washington, Seattle,
WA) for providing the founder transgenic mice for lines
2300COL
I and
440COL. We thank D. Walker, K. Pak, and A. Nesterova for technical assistance and L. Masse for the preparation of this manuscript.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants DK-38652, DK-46971, and GM-47165 and by grants from the Dept. of Veterans Affairs. K. S. Lee was supported by a grant from Yonsei University College of Medicine (Seoul, South Korea).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. Buck, Dept. of Medicine, Univ. of California, San Diego, VAMC, 9-111D, 3350 La Jolla Village Dr., San Diego, CA 92161.
Received 31 July 1998; accepted in final form 23 September 1998.
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