Department of Medicine, Veterans Affairs Medical Center, San Diego 92161; and Center for Molecular Genetics, University of California, San Diego, California 92037
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ABSTRACT |
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Expression of
-smooth muscle actin (
-SMA) defines the phenotype of activated
(myofibroblastic) hepatic stellate cells. These cells, but not
quiescent stellate cells, have a high level of
-SMA and c-Myb
expression, as well as increased c-Myb-binding activities to the
proximal
-SMA E box. Therefore, we analyzed the role of c-Myb in
-SMA transcription and stellate cell activation. Activated primary
rat stellate cells displayed a high expression of the
724 and
271
-SMA/luciferase (LUC) chimeric genes, which contain c-Myb
binding sites (
223/
216 bp).
-SMA/LUC minigenes with
mutation (
219/
217 bp), truncation (
224 bp), or
deletion (
191 bp) of the c-Myb binding site were not efficiently
transcribed. Transfection of wild-type c-Myb into quiescent stellate
cells, which do not express endogenous c-Myb, induced a ~10-fold
stimulation of
724
-SMA/LUC expression. Conversely,
expression of either a dominant-negative c-Myb basic domain mutant
(Cys43
Asp) or a c-Myb antisense RNA blocked
transcription from the
724
-SMA/LUC or
271
-SMA/LUC
in activated cells. Moreover, transfection of c-myb antisense,
but not sense, RNA inhibited both expression of the endogenous
-SMA
gene and stellate cell activation, whereas transfection of
c-myb stimulated
-SMA expression in quiescent stellate
cells. These findings suggest that c-Myb modulates the activation of
stellate cells and that integrity of the redox sensor Cys43
in c-Myb is required for this effect.
liver fibrosis; oxidative stress
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INTRODUCTION |
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STELLATE CELLS PLAY a key role in the pathogenesis of
hepatic fibrosis (14, 36). Although we (5) and others (13, 15) have
reported that quiescent stellate cells produce little collagen type I,
activated (myofibroblastic) stellate cells display a high level of
collagen 1(I) gene expression (13, 14, 36). Therefore, stellate cell
activation is a critical step in hepatic fibrogenesis. Studies with
primary cultures of adult rat stellate cells have provided evidence
that cell type-specific growth regulatory mechanisms exist (41), but
the cell-specific factors regulating stellate cell activation have only
been partially identified (9). We found that oxidative stress is a
common and indispensable step in the cascade of molecular events
initiated by collagen type I matrix or transforming growth factor-
(TGF-
), resulting in stellate cell activation (31).
The expression of -smooth muscle actin (
-SMA) defines the
activated phenotype of stellate cells (43). In this context, we have
previously reported that c-Myb expression and/or binding activities to
an oligonucleotide including the proximal E box of the
-SMA gene are
increased in association with enhanced oxidative stress in activated
stellate cells in culture (31), in animals treated with
CCl4 (31, 32), and in patients with chronic hepatitis C
(26). These findings strongly suggest that c-Myb is a molecular mediator of oxidative stress on stellate cell activation (26, 31, 32).
Although little is known about the mechanisms that modulate c-Myb
activity, it has been suggested that oxidation of Cys43
could function as a molecular sensor for the redox state of the cell by
affecting the DNA-binding affinity of c-Myb (38). In agreement with
this hypothesis, we found that addition of purified redox protein Ref-1
(52, 53) to nuclear extracts from activated stellate cells inhibits
their binding (presumably c-Myb) to the -SMA-proximal E box (32). In
this study, we show that c-Myb plays a major role in the transcription
of the
-SMA gene in activated stellate cells. Moreover,
c-myb antisense RNA blocked the development of the
myofibroblastic phenotype and expression of the endogenous
-SMA gene
induced in stellate cells by collagen type I matrix, whereas
transfection of c-myb stimulated
-SMA expression in
quiescent stellate cells.
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METHODS |
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Cell cultures.
Stellate cells were prepared from male Sprague-Dawley rats
(400-500 g) by in situ perfusion and single-step density Nycodenz gradient (Accurate Chemical & Scientific, Westbury, NY), as described previously (5, 11, 22, 23, 25). Cells were plated on collagen type I or
EHS matrix (Matrigel; Collaborative Biomedical Products,
Bedford, MA) tissue culture dishes, according to the experimental
design, with the initial seeding of fat-storing cells at a density of 2 × 105/cm2. Matrigel's major components
are laminin, collagen IV, proteoglycans, entactin, and nidogen. It also
contains TGF-, fibroblast growth factor, and tissue plasminogen
activator. Cells were cultured under an atmosphere of 5%
CO2/95% air in tissue culture dishes using DMEM containing
100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 10% FBS
(GIBCO BRL, Gaithersburg, MD) and 10% FCS (Omega, Tarzana, CA). Medium was changed every 48 h for all conditions. 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 (2).
Greater than 95% of the cells were stellate cells. Freshly isolated
stellate cells were transfected with the mammalian vectors expressing
the protein or reporter of interest using lipofectin (GIBCO BRL), as
described by the manufacturer. To increase the transfectability of
activated cells, a transfection-enhancing reagent (Life Technologies,
Gaithersburg, MD) was added in conjunction with lipofectamine as
recommended by the manufacturer. The efficiency of transfection was
determined using the pRSV-
-galactosidase vector (7).
Immunohistochemistry.
Cells fixed with acetone and methanol (60:40) at 20°C for 20 min were immunostained as described previously (6, 7, 23). Monoclonal
-SMA and anti-rabbit
-galactosidase antibodies were obtained from
Sigma (St. Louis, MO) and Cappel (Durham, NC). Oregon green and Texas
red secondary fluorochromes were obtained from Molecular Probes
(Eugene, OR). Fluorescent labels were visualized using a triple-channel
Nikon microscope as described previously (6, 7, 23, 25). The number of
SMA-positive cells was expressed as a percentage of total
transfected cells. At least 100 transfected cells were analyzed per
experimental point, and a minimum of two observers analyzed each
immunohistochemical experiment independently, as described previously
(7, 25). Negative control samples were processed in parallel under the
same conditions but with omission of the first antibody. Hoechst 33342 was used as a nuclear counterstain.
Statistical analysis. All results are expressed as means ± SE. Student's t-test was used to evaluate differences of the means between groups, with P < 0.05 considered significant.
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RESULTS |
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To assess the role of c-Myb on the transcription from the -SMA gene,
we first characterized the cis-regulatory region within the
-SMA gene 5' flanking sequences that is necessary for high expression of the
-SMA in activated stellate cells. Primary rat stellate cells cultured on a collagen type I matrix became activated (23, 25, 31) and were transfected on day 4 with
-SMA
chimeric reporter minigenes containing
724 bp,
271 bp,
230 bp,
271/
230 bp,
224 bp, or
191
bp of the rat
-SMA promoter (4) and expressing LUC (Fig.
1A). To obtain optimal reporter
expression, cells were harvested 48 h after transfection. In these
activated stellate cells, transcription from
-SMA/LUC reporter genes
was very high when the chimeric gene contained
724 bp or
271 bp of the 5' flanking region of the
-SMA promoter
[including both E boxes and the TGTTTATC motif (distal to
224 bp); Fig. 1B]. Truncation of the region
containing the distal
-SMA E box to
224 bp (including the
proximal E box) or
191 bp (including the CArG A and
B boxes) eliminated the transcription inducibility of the
-SMA gene
(Fig. 1B), which is characteristic of the early phases of
stellate cell activation (9, 43). The core binding for c-Myb, CATAAGCA (
223/
216), which is distal to the proximal E box, is
disrupted with the truncation at
224 bp. However, conservation
of the c-Myb (and other cooperative transcription factors) cognate DNA
with the truncation at
230 bp (only an additional 6 bp) leads to
a much higher
-SMA/LUC expression (Fig. 1B). Moreover,
mutation of the c-Myb binding site (A-219
T; C-217
T)
(Fig. 1A) markedly inhibited transcription from the
271
-SMA/LUC reporter gene (Fig. 1B). Furthermore, a
271
bp/
230 bp cis element (271/230
-SMA/LUC), containing
the distal E box but neither the c-Myb-binding region nor
the proximal E box, had only background LUC expression. In addition to
a role for the c-Myb cis element in the transcription from the
-SMA promoter, the TGTTTATC motif (
233 to
226),
immediately distal to the c-Myb binding site, also contributes to the
-SMA transcription in activated stellate cells. Mutation of this
motif (
TTATC) (
271 bp
[232/231 mut]
-SMA/LUC) markedly decreases the
expression of
-SMA/LUC (Fig. 1B).
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Because c-Myb expression is induced during the early stages of stellate
cell activation and c-Myb binds with high affinity to an
oligonucleotide including the proximal E box (GCAGCT 218 to
213 bp) of the
-SMA promoter
(5'-GAT
GCTGAACTGCC-3') (32), we
investigated whether c-Myb is capable of stimulating transcription from
the
-SMA promoter in quiescent stellate cells. Day 4 primary
rat stellate cells, growing on an EHS matrix to prevent
their spontaneous activation and the expression of the endogenous c-Myb
(11, 25, 31), were transfected with a vector expressing wild-type
c-Myb. Nuclear expression of c-Myb as determined by
immunofluorescence (31) (data not shown) was sufficient to increase the
basal transcription from the cotransfected
724
-SMA/LUC reporter gene by ~10-fold (216 ± 52 vs. 2,376 ± 850 U/mg protein; P < 0.05) (Fig. 2). In addition,
a c-Myb basic domain mutant (Cys43
Asp) that binds
cognate DNA with reduced affinity (38) behaved as a dominant negative
when expressed in activated stellate cells (growing on a collagen type
I matrix), markedly decreasing the
724
-SMA/LUC reporter
expression (2,670 ± 29 vs. 320 ± 130 U/mg protein; P < 0.05) (Fig. 3A).
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These results suggest that c-Myb is both sufficient and necessary to
stimulate, perhaps in concert with other transcription activators (21,
28, 31), a high level of transcription from the -SMA promoter in
activated stellate cells. We have previously reported that
c-myb antisense oligonucleotides inhibited the activation of
stellate cells induced by TGF-
in a conditioned medium (31). However, Burgess et al. (8) suggested that the antiproliferative activity of antisense c-myb-specific oligonucleotides, at least in smooth muscle cells (SMC), is not due to a hybridization-dependent antisense mechanism. Therefore, to circumvent this confounding issue
(8), we transfected day 4 activated primary rat stellate cells,
growing on a collagen type I matrix, with a vector expressing antisense
c-myb RNA and assessed the expression of the
724
-SMA/LUC reporter. As we reported previously for c-myb
antisense oligonucleotides (31), c-myb antisense RNA also
blocked c-Myb expression. In agreement with the results obtained by
expressing a dominant-negative c-myb mutant (Fig. 3A),
expression of the antisense, but not sense, c-myb RNA inhibited
by approximately fivefold the
724
-SMA/LUC reporter activity
(Fig. 3A) and by approximately threefold expression from the
271
-SMA/LUC (data not shown). These experiments strongly support the notion that c-Myb is required for optimal expression of the
-SMA gene. Furthermore, these effects were selective for the
724 bp and
271 bp 5' flanking sequences, since
c-myb antisense RNA did not modify the (already modest)
expression of the
224
-SMA/LUC reporter (data not shown). As
expected, overexpression of c-myb in activated stellate cells,
stimulated (approximately threefold) transcription from the
271
-SMA/LUC minigene but not from the same construct with point
mutations of the c-Myb binding site within the proximal E box
(
271 [
219/
217 mut]
-SMA/LUC) (Fig.
3B).
In addition, we assessed whether expression of the c-myb
antisense RNA could also inhibit expression of the endogenous -SMA gene in stellate cells and, therefore, their activation (9, 13, 43).
Day 0 stellate cells growing on a collagen type I matrix were
transfected with vectors expressing
-galactosidase with either pcDNA
(control) or c-myb antisense RNA. On day 4, cells were
fixed and analyzed in a triple-channel microscope by immunohistochemistry with specific antibodies against
-galactosidase and
-SMA, as described previously (23, 31, 32). Nuclei were stained
in blue with Hoechst 33342. As expected, control stellate cells
transfected with pcDNA (together with the transfection indicator
-galactosidase; in red) became activated, judged by their
myofibroblastic phenotype and their expression of
-SMA (in green)
(Fig. 4A,
-galactosidase). The
transfected
-galactosidase is shown in yellow because of the
superimposition of the green
-SMA over the red
-galactosidase.
Approximately 70% of the control cells transfected with pcDNA adopted
an activated phenotype and expressed
-SMA (Fig. 4B). In
contrast, the majority of cells expressing the c-myb antisense
RNA (together with the cotransfected
-galactosidase) had the
phenotype of quiescent stellate cells (nonmyofibroblastic; in red)
(Fig. 4A). More importantly, expression of c-myb
antisense RNA was sufficient to block the induction of the endogenous
-SMA gene, which is expected in day 4 primary stellate cells
growing on a collagen type I matrix (13, 22, 25, 31, 32). Only <10%
of the cells transfected with c-myb antisense RNA expressed
-SMA and had the activated phenotype (Fig. 4B). Omission of
the first antibody resulted in negative immunofluorescence for both
-SMA- and
-galactosidase-positive samples, as described
previously (6, 7, 23, 25, 31).
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In addition, day 1 quiescent stellate cells growing on EHS were
transfected with the indicator green fluorescent protein (in green) and
either control -galactosidase or c-myb. On day 4, cells were stained for
-SMA (in red). Although only ~2% of
control cells expressed
-SMA (Fig. 5),
~40% of cells transfected with c-myb were
-SMA-positive
(Fig. 5). However, unlike activated stellate cells growing on collagen
type I (Fig. 4B), c-myb-transfected cells growing on
EHS displayed a diffuse rather than a fibrillar
-SMA phenotype.
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DISCUSSION |
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Expression of -SMA and a myofibroblastic phenotype defines stellate
cell activation (43). In this study, we have characterized some of the
molecular mechanisms involved in the activation of stellate cells, an
important step in hepatic fibrogenesis (13, 14).
We have previously reported that quiescent primary rat stellate cells,
cultured on an EHS matrix, are activated by the generation of free
radicals using ascorbic acid/FeSO4 as well as by
malondialdehyde, a product of lipid peroxidation (31). In addition,
enhanced hepatic oxidative stress in animals treated with
CCl4 (31, 32) or in patients with chronic hepatitis C (26)
was associated with stellate cell activation. Complementary results
supporting the role of oxidative stress in stellate cell activation
include the finding that stellate cell activation induced by collagen type I matrix or TGF- can be blocked by antioxidants, such as D-
-tocopherol or butylated hydroxytoluene (31).
Furthermore, a pilot study suggests that D-
-tocopherol
can prevent stellate cell activation in patients with chronic hepatitis
C (26).
Several studies indicate that c-Myb plays an important role in cell
differentiation and proliferation (34, 35). For example, regulation of
c-myb expression is critical for the growth and differentiation
of the progeny of hematopoietic cells (1, 17, 50). c-Myb protein binds
to a consensus cognate DNA (16) through three homeo domain-like regions
(44) and activates the transcription of target genes (3, 29, 51). The
molecular mechanism responsible for stellate cell -SMA expression
and activation in primary cultures growing on collagen
type I (25, 31) in animals treated with CCl4
(22, 31) and in patients with chronic hepatitis C (26) seems to be
associated with increased c-myb expression (26, 31, 32) and
binding of nuclear proteins to the proximal
-SMA E box (31, 32).
The appropriate expression of the -SMA gene requires the interaction
of cell type-specific sequences within the promoter and transcriptional
factors (18-20, 37, 45, 49). Although in SMC
125 bp of the
5' flanking region are sufficient to confer high expression of
the
-SMA gene, at least
271 bp are required in skeletal
myotubes (45), which also express
-SMA. Critical cis-acting
elements within the
125 bp of the
-SMA gene in SMC include
the CArG boxes, which bind serum-response factors (45) and are
necessary for ANG II inducibility (18). A mesenchymal transcription
factor, MHox, mediates the ANG II stimulation of
-SMA expression
(20). In addition, stimulation of
-SMA gene expression by TGF-
in
SMC requires the interaction of the CArG A (
62 bp) and B
(
112 bp) boxes, together with the TGF-
control element
(
42 bp) (19).
Here we demonstrated that, unlike in SMC, high-level transcription of
-SMA/LUC chimeric genes in activated stellate cells requires more
than
224 bp within its 5' flanking region. This
271
bp 5' region includes the E boxes, the TGTTTATC motif, and a
c-Myb binding site (37, 45). In contrast, the presence of
224 bp
(including the proximal E box as well as the CArG boxes) is not
sufficient for efficient transcription from the
-SMA promoter in
activated stellate cells. Studies including truncations, deletions, and
mutations suggest which are the relevant cis elements within the
724 bp of the 5' region of the
-SMA gene in
activated stellate cells. Most likely, the
224 bp truncation
disrupts the c-Myb binding site (
223/
216) because
addition of only 6 bp (
230
-SMA/LUC) restores, to a
substantial extent, transcription from the
-SMA promoter. Indeed,
mutation of the c-Myb binding site (
219/
217 bp mut),
within the proximal E box, markedly inhibits expression from the
271
-SMA/LUC reporter gene in activated stellate cells. We
found that the TGTTTATC motif also plays a role in
-SMA
transcription in activated primary rat stellate cells because a
mutation of this site (T
TTATC) impairs expression of the
-SMA/LUC. Constructs displaying the distal E box and adjacent
cis elements (
271/
230
-SMA/LUC) had only a
background level of expression.
Transfection of a vector expressing wild-type c-Myb in quiescent
stellate cells, which express negligible quantities of nuclear c-Myb
(31), was sufficient to induce a ~10-fold stimulation of the
724
-SMA/LUC reporter gene. As expected, transfection of
c-myb stimulated expression of the
271
-SMA/LUC but
not of the
271 [
219/
217 mut]
-SMA/LUC. Conversely, expression of a c-myb antisense RNA in
activated stellate cells prevented, to a substantial extent,
transcription from the
724
-SMA/LUC and
271
-SMA/LUC chimeric genes. These studies indicate that c-Myb plays an
important role in the transcription of the
-SMA gene in stellate
cells. In chicken,
-SMA gene expression also involves a conserved
sequence motif at
225 to
233 bp (TGTTTATC) (37), which is
included in the
724
-SMA/LUC and
271
-SMA/LUC
constructs that are fully expressed in activated stellate cells.
Therefore, it is conceivable that the c-Myb binding site
(
223/
216) interacts with the adjacent TGTTTATC motif
through the formation of a transcriptional unit involving c-Myb. Indeed
c-Myb is able to cooperate with other transcription factors, including
CCAAT/enhancer binding protein-
(28), which transactivates the
collagen
1(I) enhancer (21).
As reported for SMC and myotubes (45), -SMA-expressing cells, a
construct containing more than
547 bp of the
-SMA promoter was also transcriptionally active in myofibroblastic
stellate cells growing on collagen type I matrix. However, the
724
-SMA/LUC construct was inactive in quiescent stellate
cells growing on an EHS matrix. It remains to be determined whether
repression of the
-SMA gene occurs in quiescent stellate cells
through the MCAT motif as reported for other cell types (49).
Although little is known about the mechanisms that modulate
c-myb expression, it has been suggested that oxidation of
Cys43 could function as a molecular sensor for the redox
state of the cell by affecting the DNA-binding affinity of c-Myb (38).
The modulation of AP-1 proteins involving oxidative
stress is mediated by the nuclear redox factor Ref-1 (52), which also
functions as a DNA repair enzyme (53). Ref-1 stimulates DNA-binding
activity of several transcription factors, including c-Myb, and may
itself be under a posttranslational control that is sensitive to the redox state of the cell (53). The redox activity of Ref-1 is mediated
through a conserved Cys amino acid motif (KCR) that is present in Fos,
c-Jun, and related proteins. In c-Myb, redox changes probably affect
the motif KQCR (which includes Cys43) within the DNA
binding domain. In agreement with this novel hypothesis (38, 53), we
reported that oxidative stress affects the DNA-binding activity and
expression of c-Myb (31). Although the molecular mechanisms remain to
be elucidated, the increased expression of c-Myb could be achieved, for
instance, by positive autoregulation of c-myb (39) through the
redox modulation of c-Myb protein (53). We found that both the reducing
agent dithiothreitol and the redox enzyme Ref-1 prevent
the binding of c-Myb in nuclear extracts of activated stellate cells to
the -SMA-proximal E box (31, 32), suggesting a redox mechanism that
may involve c-Myb, as proposed in cell-free systems (38, 52, 53).
Because of the previous suggestion that oxidative stress may modulate
-SMA-proximal E box-binding activities in activated stellate cells
through c-Myb (32), we assessed whether the c-Myb basic domain
(including Cys43) is a molecular target necessary for
activation. Day 0 stellate cells were cotransfected with a
c-Myb Asp43 mutant and the
-SMA/LUC reporter gene.
Expression of c-Myb Asp43, lacking the redox sensor
Cys43 (38), did markedly reduce transcription from the
724
-SMA/LUC in activated stellate cells. These results
suggest that modification of Cys43 within the DNA-binding
domain of c-Myb is critical in stimulating
-SMA gene expression from
its promoter. The signal-transduction pathway targeting c-Myb
Cys43 is likely to involve either an oxidative
modification, such as an aldehyde adduct (6, 10, 24, 42), or a
nitrosylation of Cys43 (12, 48), because the
Cys43 mutation would be refractory to either pathway. In
this context, oxidative stress pathways are known to stimulate, at
least in skeletal muscle (6), liver, and brain (M. Buck, unpublished observations), nitric oxide synthase expression and activity resulting in the synthesis of NO, which interacts with superoxide to generate peroxynitrite (33, 48), a compound highly reactive with
sulfur-containing amino acids, such as Cys (12). However, NO,
apparently in the absence of oxidative stress, downregulates, at least
in rat lung fibroblasts,
-SMA expression (54).
What is the physiological relevance of c-Myb activity in the expression
of the endogenous -SMA gene during stellate cell activation? We have
previously suggested that c-Myb plays a critical role in the expression
of
-SMA, on the basis of experiments in which c-myb
antisense phosphorothioate oligonucleotides prevented
-SMA
expression and the activation of stellate cells induced by TGF-
(31). However, this argument may not be valid because the
antiproliferative activity of c-myb-specific oligonucleotides, at least in SMC, is not due to hybridization-dependent antisense mechanisms (8). Rather, a stretch of four contiguous guanosine residues, which is present in the antisense c-myb used by us
(31) and others (46, 47), may be responsible for the sequence-specific but nonantisense antiproliferative effects of these oligonucleotides.
Because proliferation and activation of stellate cells are usually
linked (23, 25, 31, 32) and c-Myb stimulates both the proliferation and
activation of these cells (31), we studied the effects of c-myb
antisense RNA on stellate cells. Our results indicate that expression
of c-myb antisense RNA markedly inhibits stellate cell
activation (and presumably proliferation), whereas transfection of
c-myb into quiescent stellate cells stimulates -SMA
expression. It is conceivable that the antagonistic effects of
phosphorylated Ser133 CREB and oxidatively
modified c-Myb Cys43 on the stellate cell cycle (25, 31)
are mediated through cyclin/cdk repression/derepression. For example,
CREB-binding protein (27) is known to bind preferentially to the
oncoprotein c-Myb or to CREB, depending on the cell cycle state of the
cell (30, 40).
In summary, this study, in conjunction with our previous results (26,
31, 32), strongly suggests that c-Myb is a key transcription activator
of the -SMA gene in activated stellate cells by interacting with the
proximal E box (and possibly other distal cis elements) and
behaving as a redox sensor through its DNA-binding domain. These
findings may facilitate development of therapeutic approaches to
prevent stellate cell activation in chronic liver diseases.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. G. K. Owens and C. A. McNamara for
providing the genomic rat -SMA DNA,
271
-SMA, and
271 [232/231 mut]
-SMA and to Dr. O. S. Gabrielsen for the c-Myb and c-Myb Asp43 mutant constructs.
We thank Tao Li for technical assistance and Amy King for the
preparation of this manuscript.
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FOOTNOTES |
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This study was supported by the National Institutes of Health Grants DK-38652, DK-46971, and GM-47165 and by grants from the Department of Veterans Affairs. D. J. Kim was supported by a grant from Il Song Foundation (South Korea), and M. Buck was supported by fellowships from the National Cancer Institute and the American Liver Foundation.
M. Buck's present address is The Salk Institute for Biological Studies, Molecular Biology and Virology Lab, P. O. Box 85800, San Diego, CA 92037.
D. J. Kim's present address is Department of Internal Medicine, Chunchon Sacred Heart Hospital, College of Medicine, Hallym University, 153 Kyo-Dong, Chunchon-Si, Kangwon-Do, 200-060 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 and other correspondence: M. Chojkier, Dept. of Medicine and Center for Molecular Genetics, Univ. of California, San Diego 9-111D, San Diego, CA 92161.
Received 3 March 1999; accepted in final form 3 November 1999.
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