Alcohol Research and Treatment Center, Bronx Veterans Affairs Medical Center and Mount Sinai School of Medicine, Bronx, New York 10468
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dilinoleoylphosphatidylcholine
(DLPC), the active component of polyenylphosphatidylcholine extracted
from soybeans, decreases collagen accumulation induced by TGF-1 in
cultured hepatic stellate cells (HSCs). Because DLPC exerts antioxidant
effects and TGF-
1 generates oxidative stress, we evaluated whether
the antifibrogenic effect of DLPC is linked to its antioxidant action.
In passage 1 culture of rat HSCs, TGF-
1 induced a
concentration-dependent increase in
procollagen-
1(I) mRNA levels and enhanced
intracellular H2O2 and superoxide anion
formation and lipid peroxidation but decreased GSH levels. These
changes were prevented by DLPC. Upregulation of collagen mRNA by
TGF-
1 was likewise inhibited by catalase and p38 MAPK inhibitor
SB-203580, suggesting involvement of H2O2 and
p38 MAPK signaling in this process. TGF-
1 or addition of H2O2 to HSCs activated p38 MAPK with a rise in
procollagen mRNA level; these changes were blocked by catalase and
SB-203580 and likewise by DLPC.
-Smooth muscle actin abundance in
HSCs was not altered by TGF-
1 treatment (with or without DLPC),
indicating that downregulation of procollagen mRNA by DLPC was not due
to alteration in HSC activation. These results demonstrate that DLPC prevents TGF-
1-induced increase in collagen mRNA by inhibiting generation of oxidative stress and associated
H2O2-dependent p38 MAPK activation, which
explains its antifibrogenic effect. DLPC, an innocuous phospholipid,
may be considered for prevention and treatment of liver fibrosis.
dilinoleoylphosphatidylcholine; oxidative stress; antioxidant; catalase; p38 inhibitor SB-203580
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
POLYENYLPHOSPHATIDYLCHOLINE
(PPC), a mixture of polyunsaturated phosphatidylcholines extracted from
soybeans, protects against alcoholic fibrosis and cirrhosis in baboons
(38) and attenuates fibrosis induced by carbon
tetrachloride or heterologous albumin in rats (40). The
protection afforded by PPC against fibrosis was associated with
decreased transformation of hepatic stellate cells (HSCs) into
myofibroblast-like cells (41), implying that PPC may have
a direct inhibitory effect on HSC fibrogenesis. Indeed, when PPC was
tested for its antifibrogenic effect in cultured rat HSCs, it decreased
acetaldehyde-stimulated collagen accumulation in the culture media
(36). The inhibition was attributable, in part, to the
stimulation of collagenase activity by its active component
dilinoleoylphosphatidylcholine (DLPC), which constitutes 45-50%
of the PPC extract (38). DLPC was also shown to mimic the
inhibitory effect of PPC on platelet-derived growth factor stimulation
of HSC proliferation in passaged culture cells, possibly through
inhibition of the mitogen intracellular signaling cascade (5). Furthermore, we have recently reported that the
addition of DLPC to cultured rat HSCs resulted in downregulation of
mRNA levels of procollagen-1(I) and of the tissue
inhibitor of metalloproteinase (TIMP)-1 induced by TGF-
1, leading to
lesser accumulation of collagen type 1 in the culture media
(6). The mechanism by which DLPC opposes the induction of
fibrogenesis by TGF-
1 has not yet been elucidated.
TGF-1 is a potent profibrogenic cytokine that mediates tissue matrix
homeostasis in cultured HSCs, particularly in the later phase of HSC
activation (32, 63). Its fibrogenic action has been shown
to be mediated, in part, by H2O2, which is
increased by the cytokine (20). Exogenous addition of
H2O2 to HSCs in culture elicited an
upregulation of procollagen-
1(I) mRNA, mimicking that of
TGF-
1 treatment. Furthermore, both TGF-
1- and
H2O2-induced collagen gene expression were
blocked by catalase, an H2O2 scavenger, and by
the chemical antioxidant pyrrolidine thiocarbamate. These data are
consistent with the role of reactive oxygen species (ROS) in the
promotion of HSC fibrogenesis (8, 20, 35, 47, 51, 61).
In several cell types, including HSCs, H2O2
acts as a second messenger in TGF-1 signaling cascades (15,
20, 48), which involve, in addition to the SMAD group of
proteins (26, 44), the MAPKs. MAPKs are important
signal-transducing enzymes, unique to eukaryotes, that regulate many
cellular functions, including gene expression, immune response, cell
proliferation, apoptosis, and response to oxidative stress.
Four subgroups of the MAPK family have been identified, which include
ERK1/2, JNKs, p38 proteins (p38
/
/
/
), and ERK5 (reviewed in
Refs. 10, 14, 50). Of these, the
p38 MAPK signaling pathway is also involved in the induction of
procollagen-
1(I) mRNA by TGF-
1 in rat glomerular mesangial cells (11), since inhibition of p38 activation
with SB-203580, the selective pharmacological p38 inhibitor
(13), prevents the induction of collagen mRNA. In human
gingival fibroblasts, Ravanti et al. (55) showed that p38
activation is required for the induction of collagenase-3 expression by
TGF-
1. There is also evidence that, in the cytokine-induced
signaling pathway, ROS participate in the activation of p38 (reviewed
in Ref. 50). Indeed, Clerk et al. (12) found
that H2O2 directly activates p38 in rat hearts
during ischemia-reperfusion, which contributes to myocyte
hypertrophy. However, the involvement of oxidative stress-dependent p38
MAPK activation, leading to fibrogenesis triggered by TGF-
1 in HSCs,
has not been evaluated. Understanding this oxidant-based signaling
mechanism provides an opportunity for the evaluation of the therapeutic
efficacy of antioxidant agents against liver fibrosis.
Since DLPC, like PPC, has antioxidant properties (1, 46),
the present study was undertaken to evaluate its antioxidant effects
against the generation of oxidative stress and the induction of
procollagen mRNA by TGF-1 in culture-activated HSCs and to assess
its putative antioxidant action against
H2O2-mediated activation of p38 and the
associated enhanced induction of collagen mRNA. The effects of DLPC
were studied in parallel with those of catalase and the p38 inhibitor
SB-203580.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of HSCs
Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA), weighing 350-600 g and fed Purina chow and water ad libitum, were used for isolation of HSCs. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals." Nonparenchymal cells were isolated from the liver by sequential in situ perfusion with collagenase and protease as described before (45). HSCs were separated from other nonparenchymal cells over a discontinuous two-layer Nycodenz gradient (11.4 and 17%; Sigma, St. Louis, MO) prepared essentially as described by Knook et al. (33). Isolated HSCs were seeded onto plastic tissue culture flasks at 0.9-1.1 × 106 cells/ml in DMEM containing 10% FCS, 2 mM L-glutamine, 100 IU penicillin, and 100 mg/ml streptomycin (GIBCO BRL, Rockville, MD). HSCs were incubated at 37°C in a 5% CO2-air humidified atmosphere. The medium was changed 24 h after plating and 48 h thereafter. Cells were grown to subconfluence and then trypsinized. In all experiments, these cells were subcultured in DMEM supplemented with 10% FCS and the antibiotics and used 3 days later (as passage 1 HSCs). By phase contrast and immunocytochemical microscopy, these cells revealed an activated HSC phenotype as indicated by positive staining forTreatment of HSCs
Passage 1 HSCs were incubated with serum-free DMEM containing 0.05% bovine albumin (vehicle control), TGF-RNA extraction and Northern blot analysis.
HSCs were incubated with the above reagents for 24 h, and the
cellular RNA was extracted with acid guanidinium
isothiocyanate-phenol-chloroform and used for Northern blot analysis
according to a standard technique (42). A cDNA probe for
rat procollagen-1(I) or
-actin (American Type Culture
Collection, Manassas, VA) was labeled with [32P]dCTP by
using a random priming DNA labeling kit (Amersham, Arlington Heights,
IL). Levels of mRNA were quantified by measuring the intensity of the
bands on X-ray film with the MCID image analysis system (Imaging
Research, St. Catherines, ON, Canada).
Oxidative Stress Assessment
HSCs (3 × 105) in six-well culture plates were incubated with TGF-H2O2 generation. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH2-DA; Molecular Probes, Eugene, OR) was used to measure intracellular H2O2 generation by the method of Carter et al. (7). DCFH2-DA is freely permeable across cell membranes and is incorporated into hydrophobic lipid regions of the cell. H2O2 produced by the cell oxidizes DCFH2-DA to 2',7'-dichlorofluorescein (DCF), the fluorescence of which is proportional to the H2O2 produced. DCFH2-DA was added to the culture at a final concentration of 20 µM, and DCF fluorescence was measured 30 min thereafter by flow cytometry with a FACScan cytometer (BD Immunocytometry Systems, San Jose, CA) at 488 nm for excitation and 525 for emission. Background reading from cells incubated without the probe was subtracted, and data were analyzed with Cell Quest software.
Superoxide anion generation. Hydroethidine was used to measure superoxide anion generation by the method described by Carter et al. (7). Hydroethidine is freely permeable to cells and can be directly oxidized to ethidium bromide by superoxide anion produced by the cell. The loss of fluorescence in the cells is proportional to the superoxide anion generated. Hydroethidine (Molecular Probes) was added to the culture at a final concentration of 10 µM, and the fluorescence was measured 30 min thereafter by spectrofluorometry at 352 nm for excitation and 434 nm for emission.
Lipid peroxidation detection. Lipid peroxidation was measured according to the method described by Kuypers et al. (34). Cis-parinaric acid (Molecular Probes) is a fluorescent polyunsaturated fatty acid that is incorporated into cellular membranes. Subsequent to peroxidative stress, cis-parinaric acid is degraded, resulting in decreased intensity. Hence the loss of fluorescence is proportional to lipid peroxidation. Cis-parinaric acid was added to the culture at a final concentration of 5 µM, and the fluorescence was measured by spectrofluorometry at 325 nm for excitation and 413 nm for emission.
Measurement of GSH. GSH levels were determined by a Glutathione assay kit (Cayman, Ann Arbor, MI) according to the manufacturer's instructions.
p38 MAPK (Thr180/Tyr182) Phosphorylation Assay
Phosphorylation of p38 was assayed by using the components provided in the PhosphoPlus p38 MAPK antibody kit obtained from Cell Signaling Technology (Beverly, MA). HSCs (1 × 106) grown on 100-mm culture dishes were treated with the reagents (described above) for 0.5, 2, and 24 h. The cells were then lysed by adding 100 µl SDS sample buffer containing 62.5 mM Tris · HCl (pH 6.5), 2% wt/vol SDS, 10% glycerol, 50 mM DTT, and 0.1% wt/vol bromphenol blue. These cells were immediately scraped off the plates and transferred to microcentrifuge tubes and kept on ice. The content was sonicated for 2 s to shear the DNA and reduce the sample viscosity. A 20-µl sample (20 µg protein) was boiled for 5 min, cooled on ice, and then centrifuged for 5 min. The protein was resolved on a 12% SDS-PAGE gel and electroblotted onto a nitrocellulose membrane. After being washed and blocked with blocking buffer, the membrane was incubated with phospho-p38 (Thr180/Tyr182) rabbit polyclonal antibody, which detected p38 only when activated by dual phosphorylation at Thr180 and Tyr182, overnight at 4°C. The antibody was diluted at 1:1,000. After being washed, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,000) and HRP-conjugated anti-biotin antibody (1:1,000) to detect the biotinylated protein markers. As a control for protein loading, the blot was subjected to immunoblotting for the corresponding nonphosphorylated p38 with anti-p38 antibody, which detected total p38. Immunoreactive proteins were visualized by the LumiGLO chemiluminescent reagents and then exposed to X-ray film. Signal intensities were quantified with MCID.p38 MAPK Activity Assay
The kinase activity of p38 was assayed by detection of activating transcription factor (ATF)-2 phosphorylation by using the p38 MAP kinase assay kit provided by Cell Signaling Technology. HSCs (1 × 106 cells) on culture dishes were lysed in a buffer containing 20 mM Tris · HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na-pyrophosphate, 1 mMWestern Blot Analysis of -SMA
Protein Determination
HSC lysate protein content was determined using the BCA protein assay kit from Pierce (Rockford, IL).Cell viability assay. The viability of HSCs after treatment with the various reagents was determined by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay according to the method described by Imamura et al. (27) and Martinou et al. (43).
Statistics
Data are reported as means ± SE. Statistical analysis was performed by ANOVA followed by Student-Newman-Keuls tests for multiple comparisons between treatment groups using Instat (v. 3.01) and Sigma Stat (v. 2.0) software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Induction of Procollagen-1(I) mRNA by TGF-
1:
Concentration Effect and Inhibition by DLPC
|
Generation of Oxidative Stress by TGF-1 and Inhibition by
DLPC
|
Effects of Catalase and SB-203580 on TGF-1-Induced
Procollagen-
1(I) mRNA
|
Phosphorylation of the p38 MAPK by TGF-1: Effects of
Concentration and Time
|
Effects of Catalase, SB-203580, and DLPC on TGF-1-Induced
p38 MAPK Phosphorylation and its Kinase Activity
|
|
H2O2-Dependent Induction of
Procollagen-1(I) mRNA and its Inhibition by
Catalase, SB-2035880, and DLPC
|
Effect of H2O2 on p38 MAPK Phosphorylation and its Kinase Activity: Inhibition by Catalase, SB-203580, and DLPC
The capacity of H2O2 to activate the p38 MAPK in HSCs was evaluated. When the oxidant was added to the HSCs in culture, a threefold increase in p38 phosphorylation and a fourfold increase in the kinase activity were found after 2 h (Fig. 8). Catalase and SB-203580 completely blocked the stimulation elicited by H2O2. The inhibition by catalase and the p38 inhibitor was mimicked by DLPC, further substantiating DLPC's involvement in the modulation of the p38 signaling by H2O2.
|
-SMA and Cell Viability After TGF-
1 and DLPC Treatment
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study revealed that DLPC, which is the active
component of PPC extracted from soybeans, prevents the induction of
procollagen-1(I) mRNA by TGF-
1 in cultured rat HSCs.
The inhibitory effect of DLPC on collagen mRNA induction is
attributable to its antioxidant action against the oxidative stress
generated by TGF-
1 and the associated
H2O2-dependent activation of p38 MAPK
signaling, leading to downregulation of collagen mRNA. DLPC mimics the
action of catalase, which scavenges H2O2, and
that of SB-203580, which blocks p38 activation, and is as effective as
these mediators against the induction of collagen mRNA elicited by
TGF-
1. These findings explain the DLPC antifibrogenic action that is
mediated by its antioxidant property.
We have previously described three major regulatory actions of TGF-1
on collagen production by cultured HSCs (6). These include
upregulation of mRNA levels of collagen type I and of TIMP-1, with
increased concentration of the corresponding proteins in the presence
of unchanged matrix metalloproteinase (MMP)-13. As a consequence,
collagen accumulates in the culture media. DLPC exerts its
antifibrogenic effect against TGF-
1 by decreasing not only mRNA
levels of procollagen but also that of TIMP-1, without affecting MMP-13
mRNA levels; this leads to lesser collagen accumulation in the culture
media. Furthermore, the specificity of the antifibrogenic property of DLPC has been verified by comparing its action with that of palmitoyllinoleoylphosphatidylcholine, the second
most abundant component of the PPC extract (38), which had
no effects on collagen synthesis and its accumulation in cultured HSCs
(6, 53).
In the present study, we have identified a novel antifibrogenic action
of DLPC. The oxidative stress generated by TGF-1 is a general
property of the cytokine, because it induces ROS formation not only in
HSCs (15, 20) but in other cell types as well, including
hepatocytes (30), lung fibroblasts (62), and
osteoblasts (49). ROS are potent mediators of fibrogenesis
in HSCs (9, 20, 35, 47, 51, 61). In vivo studies showed
that PPC decreases oxidative stress induced by alcohol consumption in
baboons (37) and prevents lipid peroxidation induced by
CCl4 in rats (1). DLPC was also found to
prevent oxidization of human low-density lipoproteins
(46). We now demonstrate that DLPC prevents the generation
of H2O2 and superoxide anion, prevents lipid
peroxidation, and restores GSH levels in HSCs after TGF-
1 treatment,
documenting its antioxidant action.
In conjunction with its blocking H2O2
accumulation generated by TGF-1 in HSCs, DLPC inhibits
H2O2-mediated phosphorylation of p38 MAPK. This
is associated with a reduction in its kinase activity, as
revealed by the decreased phosphorylation of the transcription factor
ATF-2, a substrate of p38 (29, 39, 54), which has been
shown to be activated by the TGF-
1 signaling cascade via the p38
pathway (24, 58). The efficacy of DLPC inhibition of p38
activation mediated by H2O2 and the resulting
procollagen mRNA downregulation were found to be equal to that of
catalase or of the p38 inhibitor SB-203580. Previous studies described H2O2 as a signaling molecule for TGF-
1
induction of procollagen-
1(I) mRNA in a rat HSC line
(20) and as a link between oxidative stress and enhanced
collagen-
1(I) gene expression induced by acetaldehyde in
passaged mouse HSCs (22). Importantly, the present study
provides additional data showing that H2O2
activates the p38 signaling pathway that leads to collagen mRNA
upregulation, thereby suggesting that the p38 MAPK serves as a
molecular link between H2O2 signaling and
enhanced collagen mRNA induction by TGF-
1 in HSCs.
Our finding of the inhibition of TGF-1-induced p38 activation by
SB-203580, leading to downregulation of procollagen mRNA level, is
consistent with the observation of Chin et al. (11) in
glomerular mesangial cells, which is not unexpected because activated
HSCs and mesangial cells share common characteristics of extracellular
matrix production and vitamin A storage (4). Thus far,
there is only limited information on the role of p38 signaling in HSC
fibrogenesis. A recent study showed that inhibition of p38 by SB-203580
decreased procollagen-
1(I) mRNA level in HSCs, with a
maximal effect in early primary culture (59). Another study reported that the constitutive p38 activity was higher in activated HSCs than in quiescent cells, suggesting a role for p38 in
the activation process of HSCs (56). Since the abundance of
-SMA in passage 1 HSCs was not altered by treatment
with TGF-
1, DLPC, or both, the activation of HSCs was not a factor
involved in the mediation of the antifibrogenic and antioxidant effects of DLPC. Knittel et al. (31) also observed no changes in
the level of
-SMA in culture-activated HSCs after TGF-
1 treatment.
Our data indicate that, even in their activated phenotype, HSCs are
responsive to TGF-1 stimulation of ROS generation, activation of p38
signaling, and, ultimately, upregulation of procollagen mRNA in a
concentration-dependent manner, with a maximal 3.8-fold increase at 8 ng/ml after 24 h. This level of mRNA induction is commonly
elicited in culture-activated HSCs (3, 65) and in the HSC
line (CFSC-2G) derived from a rat CCl4-cirrhotic liver (20) induced by TGF-
1 or in mouse HSCs stimulated by
acetaldehyde, a potent fibrogenic stimulator (22, 45).
Casini et al. (9) found that human passaged HSCs responded
to TGF-
1 with a concentration-dependent increase of procollagen-I
and -III mRNA abundance and of their collagen accumulation in the
culture. Recent studies by Dooley et al. (16, 17)
described changes in the responsiveness to TGF-
1 of HSCs upon
passage in culture. Whereas HSCs in the early stage of primary culture
were responsive to TGF-
1 stimulation of collagen-
2(I)
mRNA and inhibition of HSC proliferation, activated HSCs in the later
stage of primary culture were minimally responsive to TGF-
1
treatment. The loss of responsiveness of activated HSCs to TGF-
1 was
ascribed to reduced TGF-
ligand-binding activity and diminished DNA
binding of intracellular SMAD proteins, despite the normal expression
of TGF-
receptors I and II on the cell surface. A lower
collagen gene transcriptional response to TGF-
1 in the rat CFSC-2G
HSC line compared with fetal skin fibroblasts has also been reported
(28), yet these same cells upon TGF-
treatment can
produce an amount of H2O2 sufficient to lead to a significant increase in collagen mRNA level (20). In the
present study, we have not compared the responsiveness to TGF-
1
stimulation of ROS generation between passage 1 activated
and quiescent HSCs in early primary culture. However, De Bleser et al.
(15) showed that activated HSCs generate
H2O2 as well as quiescent cells after TGF-
1
treatment. Renal mesangial cells are responsive to exogenous TGF-
1,
with ensuing fibrogenesis mediated by the p38 (11) or SMAD-transducing signaling pathway (52), even after many
passages in culture. The loss of cellular response to the cytokine may be cell type and context dependent. In the case of p38 activation stimulated by TGF-
1 in activated HSCs, as observed in the present study, the signaling mechanism is most likely involved with the TGF-
-activated kinase 1 pathway, which has been shown to activate the MAPKs (58, 64), rather than the SMAD pathway. Indeed, the data reported by Schnabl et al. (60) suggested that
the activation of ERK1/2 by TGF-
1 in HSCs is independent of SMAD signaling.
In this study, we focused on the p38 signaling pathway that is
activated by H2O2 after TGF-1 treatment and
on its inhibition by DLPC. Although the JNK pathway of the MAPKs can
also be activated in response to environmental stress and by TGF-
1
signaling (64), its involvement in the induction of
collagen mRNA by TGF-
1 is less clear, with inhibition reported in
HSCs at the early stage of primary culture and no effect at the later
stage (59). In renal mesangial cells, JNK was not
activated by TGF-
1 (11) or shown to play a role in
TGF-
-1-mediated collagen-
1(I) expression (25). The ERK1/2 MAPK has been reported to be activated by
TGF-
1 in HSCs (57), but its role in fibrogenesis was
not examined.
In response to liver injury in general and fibrogenic stimuli in
particular, HSCs are activated to proliferate (18, 21) and
to transform (23, 41) into myofibroblast-like cells with an active fibrogenic phenotype, which play a major role in the onset
and progression of liver fibrosis. Because HSCs cultured on plastic
recapitulate the biological activation process of HSCs in vivo
(19), they provide a model for the evaluation of potential therapeutic agents against liver fibrosis (66). The
present study demonstrates the usefulness of culture-activated HSCs in conjunction with TGF-1 treatment as a model for the evaluation of
the efficacy of antifibrogenic agents, in particular compounds with
potential antioxidant properties, as exemplified by DLPC.
In conclusion, our findings revealed a novel mechanism by which DLPC
prevents the induction of fibrogenesis by TGF-1 through inhibition
of the oxidative stress generated by the cytokine and the associated
H2O2-dependent activation of the p38 MAPK
signaling pathway. This effect of DLPC may explain, at least in part,
how PPC opposes fibrosis, and through this action DLPC or PPC may be
useful for the treatment of alcoholic as well as nonalcoholic liver fibrosis.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported, in part, by National Institute on Alcohol Abuse and Alcoholism Grant AA-11115, the Department of Veterans Affairs, and the Kingsbridge Research Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. S. Lieber, Alcohol and Nutrition Research Center, Veterans Affairs Medical Center, Mount Sinai School of Medicine, 130 West Kingsbridge Rd., Bronx, NY 10468 (E-mail: liebercs{at}aol.com).
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. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00128.2002
Received 2 April 2002; accepted in final form 10 July 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aleynik, SI,
Leo MA,
Aleynik MK,
and
Lieber CS.
Polyenylphosphatidylcholine prevents carbon tetrachloride-induced lipid peroxidation while it attenuates liver fibrosis.
J Hepatol
27:
554-561,
1997[ISI][Medline].
2.
Aleynik, SI,
Leo MA,
Takeshige U,
Aleynik MK,
and
Lieber CS.
Dilinoleoylphosphatidylcholine is the active antioxidant of polyenylphosphatidylcholine.
J Investig Med
47:
507-512,
1999[ISI][Medline].
3.
Armendariz-Borunda, J,
Katayama K,
and
Seyer JM.
Transcriptional mechanisms of type I collagen gene expression are differentially regulated by interleukin-1, tumor necrosis factor
, and transforming growth factor
in Ito cells.
J Biol Chem
267:
14136-14321,
1992.
4.
Bauer, P,
and
Wake K.
Mesangial cells of the lamprey, Lampetra japonica, store vitamin A.
Arch Histol Cytol
59:
71-78,
1996[ISI][Medline].
5.
Brady, LM,
Fox ES,
and
Fimmel CJ.
Polyenylphosphatidylcholine inhibits PDFG-induced proliferation in rat hepatic stellate cells.
Biochem Biophys Res Commun
248:
174-179,
1998[ISI][Medline].
6.
Cao, Q,
Mak K,
and
Lieber CS.
Dilinoleoylphosphatidylcholine (DLPC) decreases transforming growth factor-1-mediated collagen production by rat hepatic stellate cells.
J Lab Clin Med
139:
202-210,
2002[ISI][Medline].
7.
Carter, WO,
Narajanan PK,
and
Robinson JP.
Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.
J Leukoc Biol
55:
253-258,
1994[Abstract].
8.
Casini, A,
Ceni E,
Salzano R,
Biondi P,
Parola G,
Galli A,
Foschi M,
Caligiuri A,
Pinzani M,
and
Surrenti C.
Neutrophil-derived superoxide anions induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells.
Hepatology
25:
361-367,
1997[ISI][Medline].
9.
Casini, A,
Pinzani M,
Milani S,
Grappone C,
Galli G,
Jezequel AM,
Schuppan D,
Rotella CM,
and
Surrenti C.
Regulation of extracellular matrix synthesis by transforming growth factor-1 in human fat-storing cells.
Gastroenterology
105:
245-253,
1993[ISI][Medline].
10.
Chang, L,
and
Karin M.
Mammalian MAP kinase signaling cascades.
Nature
410:
37-40,
2001[ISI][Medline].
11.
Chin, BY,
Mohsenin A,
Li SX,
Choi AMK,
and
Choi ME.
Stimulation of pro-1(I) collagen by TGF-
1 in mesangial cells: role of the p38 MAPK pathway.
Am J Physiol Renal Physiol
280:
F495-F504,
2001
12.
Clerk, A,
Fuller SJ,
Michael A,
and
Sugden PH.
Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinase/c-Jun N-terminal kinases and p38-mitogen activated protein kinases) in perfused rat hearts by oxidative and other stresses.
J Biol Chem
273:
7228-7234,
1998
13.
Cuenda, A,
Rouse J,
Doza YN,
Meier R,
Cohen P,
Gallagher T,
Young PR,
and
Lee JC.
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stress and interleukin-1.
FEBS Lett
364:
229-233,
1995[ISI][Medline].
14.
Davis, RJ.
Signal transduction by the JNK group of MAP kinases.
Cell
103:
239-252,
2000[ISI][Medline].
15.
De Bleser, PJ,
Xu G,
Rombouts K,
Rogiers V,
and
Geerts A.
Glutathione levels discriminate between oxidative stress and transforming growth factor- signaling in activated hepatic stellate cells.
J Biol Chem
274:
33881-33887,
1999
16.
Dooley, S,
Delvoux B,
Lahme B,
Mangasser-Stephan K,
and
Gressner AM.
Modulation of transforming growth factor response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts.
Hepatology
31:
1094-1106,
2000[ISI][Medline].
17.
Dooley, S,
Delvoux B,
Strekert M,
Bonzel L,
Stopa M,
ten Dijke P,
and
Gressner AM.
Transforming growth factor signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vivo progression to myofibroblasts.
FEBS Lett
502:
4-10,
2001[ISI][Medline].
18.
Friedman, SL.
The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.
N Engl J Med
328:
1828-1835,
1993
19.
Friedman, SL,
Roll FJ,
Boyles J,
Arenson DM,
and
Bissel DM.
Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix.
J Biol Chem
264:
10756-10762,
1989
20.
Garcia-Trevijano, ER,
Iraburu MJ,
Fontana L,
Dominguez-Rosales JA,
Auster A,
Covarrubias-Pinedo A,
and
Rojkind M.
Transforming growth factor 1 induces the expression of
1(I) procollagen mRNA by a hydrogen peroxide-C/EBPB-dependent mechanism in rat hepatic stellate cells.
Hepatology
29:
960-970,
1999[ISI][Medline].
21.
Geerts, A,
Lazou JM,
De Bleser P,
and
Wisse E.
Tissue distribution, quantification and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver.
Hepatology
13:
1193-1202,
1991[ISI][Medline].
22.
Greenwel, P,
Dominguez-Rosales JA,
Mavi G,
Rivas-Estilla M,
and
Rojkind M.
Hydrogen peroxide: a link between acetaldehyde-elicited 1(I) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells.
Hepatology
31:
109-116,
2000[ISI][Medline].
23.
Gressner, AM,
and
Bachem MG.
Molecular mechanisms of liver fibrogenesisa homage to the role of activated fat-storing cells.
Digestion
56:
335-346,
1995[ISI][Medline].
24.
Hanafusa, H,
Ninomiya-Tsuji J,
Masuyama N,
Nishita M,
Fujisawa J,
Shibuya H,
Matsumoto K,
and
Nishida E.
Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor--induced gene expression.
J Biol Chem
274:
27161-27167,
1999
25.
Hayashida, T,
Poncelet AC,
Hubchak SC,
and
Schnaper HW.
TGF-1 activates MAP kinase in human mesangial cells: a possible role in collagen expression.
Kidney Int
56:
1710-1720,
1999[ISI][Medline].
26.
Heldin, CK,
Miyazano K,
and
ten Dijke P.
TGF- signalling from cell membrane to nucleus through SMAD proteins.
Nature
390:
465-471,
1997[ISI][Medline].
27.
Imamura, H,
Takao S,
and
Aikou T.
A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion.
Cancer Res
54:
3620-3624,
1994[Abstract].
28.
Inagaki, Y,
Mamura M,
Kanmaru Y,
Greenwel P,
Nemoto T,
Takehara K,
ten Dijke P,
and
Nakao A.
Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells.
J Cell Physiol
187:
117-123,
2001[ISI][Medline].
29.
Jiang, Y,
Chen C,
Li Z,
Guo W,
Gegner JA,
Lin S,
and
Han J.
Characterization of the structure and function of a new mitogen-activated protein kinase (p38).
J Biol Chem
271:
17920-17926,
1996
30.
Kayanoki, Y,
Fujii J,
Suzuki K,
Kawata S,
Matsuzawa Y,
and
Taniguichi N.
Suppression of antioxidative enzyme expression by transforming growth factor-1 in rat hepatocytes.
J Biol Chem
269:
15488-15492,
1994
31.
Knittel, T,
Janneck T,
Muller L,
Fellmer P,
and
Ramadori G.
Transforming growth factor 1-regulated gene expression of Ito cells.
Hepatology
24:
352-360,
1996[ISI][Medline].
32.
Knittel, T,
Mehde M,
Kobold D,
Saile B,
Dinter C,
and
Ramadori G.
Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and nonparenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1.
J Hepatol
30:
48-60,
1999[ISI][Medline].
33.
Knook, DL,
Seffelaur AM,
and
de Leeuw AM.
Fat-storing cells of the liver. Their isolation and purification.
Exp Cell Res
139:
468-472,
1982[ISI][Medline].
34.
Kuypers, FA,
van den Berg JJM,
Schalkwijk C,
Roelofsen B,
and
Op den Kamp JAF
Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation.
Biochim Biophys Acta
921:
266-274,
1987[ISI][Medline].
35.
Lee, KS,
Buck M,
Houglum K,
and
Chojkier M.
Activation of hepatic stellate cells by TGF and collagen type 1 is mediated by oxidative stress through c-myb expression.
J Clin Invest
96:
2461-2468,
1995[ISI][Medline].
36.
Li, J,
Kim CI,
Leo MA,
Mak KM,
Rojkind M,
and
Lieber CS.
Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes.
Hepatology
15:
373-381,
1992[ISI][Medline].
37.
Lieber, CS,
Leo MA,
Aleynik SI,
Aleynik MA,
and
DeCarli LM.
Polyenylphosphatidylcholine decrease alcohol-induced oxidative stress in the baboon.
Alcohol Clin Exp Res
21:
375-379,
1997[ISI][Medline].
38.
Lieber, CS,
Robins SJ,
Li J,
Decarli LM,
Mak KM,
Fasulo JM,
and
Leo MA.
Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon.
Gastroenterology
106:
152-159,
1994[ISI][Medline].
39.
Livingston, C,
Patel G,
and
Jones N.
ATF-2 contains a phosphorylation-dependent transcriptional activation domain.
EMBO J
14:
1785-1797,
1995[Abstract].
40.
Ma, X,
Zhao J,
and
Lieber CS.
Polyenylphosphatidylcholine attenuates non-alcoholic hepatic fibrosis and accelerates its regression.
J Hepatol
24:
604-613,
1996[ISI][Medline].
41.
Mak KM, Leo MA, and Lieber CS. Transformation of fat-storing cells
into transitional cells in alcoholic liver fibrosis. In: Falk
Symposium 71 on "Fat-Storing Cells and Liver Fibrosis," p.
167-179. (Florence, Italy, July 1-3, 1993)
42.
Maniatis T, Fritch EF, and Sambrook J. Gel
electrophoresis. In: Molecular Cloning: a Laboratory
Manual (1st ed.), edited by Maniatis T, Fritch EF, and
Sambrook J. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory,
1982, p. 161-175.
43.
Martinou, I,
Fernandez PA,
Missotten M,
White E,
Allet B,
Sadoul R,
and
Martinou JC.
Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.
J Cell Biol
128:
201-208,
1995[Abstract].
44.
Massague, J.
TGF signal transduction.
Annu Rev Biochem
67:
753-791,
1998[ISI][Medline].
45.
Moshage, H,
Casini A,
and
Lieber CS.
Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes.
Hepatology
12:
511-518,
1990[ISI][Medline].
46.
Navder, KP,
Baraona E,
and
Lieber CS.
Dilinoleoylphosphatidylcholine protects human low density lipoproteins against oxidation.
Atherosclerosis
152:
89-95,
2000[ISI][Medline].
47.
Nieto, N,
Friedman SL,
Greenwel P,
and
Cederbaum AI.
CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells.
Hepatology
30:
987-996,
1999[ISI][Medline].
48.
Ohba, M,
Shibanuma M,
Kuroki T,
and
Nose K.
Production of hydrogen peroxide by transforming growth factor-1 and its involvement in induction of erg-1 in mouse osteoblastic cells.
J Cell Biol
126:
1079-1088,
1994[Abstract].
49.
Oneta, CM,
Mak KM,
and
Lieber CS.
Dilinoleoylphosphatidylcholine selectively modulates lipopolysaccharide-induced Kupffer cells activation.
J Lab Clin Med
134:
446-470,
1999.
50.
Ono, K,
and
Han J.
The p38 signal transduction pathway activation and function.
Cell Signal
12:
1-13,
2000[ISI][Medline].
51.
Parola, M,
and
Robino G.
Oxidative stress-related molecules and liver fibrosis.
J Hepatol
35:
297-306,
2001[ISI][Medline].
52.
Poncelet, A,
de Caestecker M,
and
Schnaper W.
The TGF- -SMAD signaling pathway is present and functional in human mesangial cells.
Kidney Int
56:
1354-1365,
1999[ISI][Medline].
53.
Poniachek, J,
Baraona E,
Zhao J,
and
Lieber CS.
Dilinoleoylphosphatidylcholine decreases hepatic stellate cells activation.
J Lab Clin Med
133:
342-348,
1999[ISI][Medline].
54.
Raingeaud, J,
Gupta S,
Rogers JS,
Dickens M,
Han J,
Ulevitch RJ,
and
Davis RJ.
Pro-inflammatory cytokines and environmental stress causes p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.
J Biol Chem
270:
7420-7426,
1995
55.
Ravanti, L,
Häkkinen L,
Larjava H,
Sarrialho-Kere U,
Foschi M,
Han J,
and
Kähäri VM.
Transforming growth factor- induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase.
J Biol Chem
274:
37292-37300,
1999
56.
Reeves, HL,
Dack CL,
Peak M,
Burt AD,
and
Day CP.
Stress-activated protein kinases in the activation of rat hepatic stellate cells in culture.
J Hepatol
32:
465-472,
2000[ISI][Medline].
57.
Reimann, T,
Hempel U,
Krautwald S,
Axmann A,
Scheibe R,
Seidel D,
and
Wenzel KW.
Transforming growth factor-1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.
FEBS Lett
403:
57-60,
1997[ISI][Medline].
58.
Sano, Y,
Harada J,
Tashiro S,
Gotoh-Mandeville R,
Maekewa T,
and
Ishii S.
ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor- signaling.
J Biol Chem
2734:
8949-8957,
1999.
59.
Schnabl, B,
Bradham CA,
Benett BL,
Manning AM,
Stefanovic B,
and
Brenner DA.
TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.
Hepatology
34:
953-963,
2001[ISI][Medline].
60.
Schnabl, B,
Kweon YO,
Frederick JP,
Wang XF,
Rippe RA,
and
Brenner DA.
The role of Smad3 in mediating mouse hepatic stellate cell activation.
Hepatology
34:
89-100,
2001[ISI][Medline].
61.
Svegliati-Baroni, G,
D'Ambrosio L,
Ferretti G,
Casini A,
Di Sario A,
Salzano F,
Ridolfi F,
Saccomanno S,
Jezequel AM,
and
Benedetti A.
Fibrogenic effect of oxidative stress on rat hepatic stellate cells.
Hepatology
27:
720-726,
1998[ISI][Medline].
62.
Thannickal, VJ,
and
Fanburg BL.
Activation of an H2O2-generating oxidase in human lung fibroblasts by transforming growth factor 1.
J Biol Chem
270:
30334-30338,
1995
63.
Tsukamoto, H.
Cytokine regulation of hepatic stellate cells in liver fibrosis.
Alcohol Clin Exp Res
23:
911-916,
1999[ISI][Medline].
64.
Wang, W,
Zhou G,
Hu MCT,
Yao Z,
and
Tan TH.
Activation of the hematopoietic progenitor kinase-1 (HPK1)-dependent, stress-activated c-Jun N-terminal kinase (JNK) pathway by transforming growth factor (TGF-
)-activated kinase (TAK1), a kinase mediator of TGF-
signal transduction.
J Biol Chem
272:
22771-22775,
1997
65.
Weiner, FR,
Giambrone MA,
Czaja MJ,
Shah A,
Annoni G,
Takahashi S,
Eghbali M,
and
Zern MA.
Ito-cell gene expression and collagen regulation.
Hepatology
11:
111-117,
1990[ISI][Medline].
66.
Wu, J,
and
Zern MA.
Hepatic stellate cells: a target for the treatment of liver fibrosis.
J Gastroenterol
35:
666-672,
2000.