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(Received for publication, January 23,
1996; and in revised form, February 29, 1996) From the
Hepatic stellate cells become activated into myofibroblast-like
cells during the early stages of hepatic injury associated with
fibrogenesis. The subsequent dysregulation of hepatic stellate cell
collagen gene expression is a central pathogenetic step during the
development of cirrhosis. The cytoplasmic Raf and mitogen-activated
protein (MAPK) kinases were found to differentially regulate
The detailed cytoplasmic signaling involved in the regulation of
key structural and/or disease-related genes is poorly understood. This
information would be useful to (i) identify potential targets for
therapeutic intervention and (ii) identify unappreciated DNA-binding
domains involved in gene regulation, as well as to link these domains
to potential upstream regulators. Previous studies have suggested that
Ras overexpression leads to a decrease in type I collagen gene
expression(1) . This implies that cytoplasmic kinase cascades
may be involved in the regulation of the collagen gene. This gene has
physiologic significance as it plays a major role in embryogenesis. In
addition, its abnormal expression during fibrogenesis is a major
feature of fibrotic liver, kidney, and lung disease. Recent studies
have described several kinase cascades that can link Ras to
transcription factor
activation(2, 3, 4, 5, 6, 7) .
A dominant pathway that is often involved utilizes the Ras We evaluated the role of the Ras-Raf-MAPK cascade during collagen
gene expression using cultured hepatic stellate cells (HSC), the major
collagen-producing effector cell responsible for hepatic fibrogenesis (16, 17, 18, 19, 20, 21) .
This system utilizes activated early passage cells, which recapitulate
many features of the diseased stellate cell in
vivo.(16, 17, 18, 19, 20, 21) .
This model can serve as a paradigm of the enhanced collagen gene
expression, which occurs in vivo during liver injury and
fibrogenesis(16, 17, 18, 19, 20, 21) .
Dominant negative inhibitory mutants were used to specifically consider
the roles of Ras, Raf, and MAPK. Overexpressing activated constructs
were avoided. Oncogenic forms of ras and raf are not
involved in hepatic fibrogenesis. In addition, these constructs may
abnormally stimulate a pathway with little physiologic relevance(4, 5, 6, 7, 20, 21) . It was found that blockage of Ras or Raf activity led to an increase
in collagen gene expression. This is consistent with the Ras
overexpression studies previously mentioned. Surprisingly, however,
blockage of MAPK activity decreased collagen gene expression. When both
Raf and MAPK activity were simultaneously blocked, collagen gene
expression was decreased. These data suggest a branch point between Ras
To evaluate the requirement for
TAE binding in vivo, transfections were performed as above
with varying concentrations of double-stranded oligonucleotides (sites
-1625 to -1615 bp in the 5`-UTR of the
Similar wild type and mutated TAEs have been shown previously to
selectively disrupt TGF
Figure 1:
Dominant negative raf and MAPK alter intcolCAT expression. A, dominant negative raf increases intcolCAT, while dominant negative MAPK suppresses intcolCAT. Hepatic stellate cells were cotransfected
with intcolCAT (plasmid -3.6/1.6, see Fig. 2) (0.5
µg/well) and either 1.0 µg of pMNC, dominant negative raf (301-1 plasmid), or dominant negative MAPK (pCMVp41(Ala-54/55)
Figure 2:
Dominant negative MAPK-induced
suppression requires the NF-1 site in footprint 1, and not the TAE.
HSCs were cotransfected with either empty vector pMNC or dominant
negative MAPK plasmid and equivalent amounts of colCAT
reporters, as described in Fig. 1. The various colCAT reporters
are labeled and schematically depicted on the left. The
relative amount of colCAT expression is displayed on the right (
Using the
same approach, the dn-raf-sensitive region was localized to a
different upstream region (Fig. 3A). Sequential
deletion analysis revealed that the 1st intron is not required, but a
region between -1.7 kb and -1.3 kb is needed. When this
region was placed adjacent to the -400 bp region of the 5`-UTR,
the previously unresponsive -400 bp-containing plasmid
(-0.4/1.6) regained its sensitivity to dn-raf stimulation(8, 30) . Previous studies in other
cell systems have suggested that this -1.7 to -1.3 kb
region contains significant DNA binding activity at the -1.62 kb
region, termed the TAE, a region previously shown to be required for
TGF
Figure 3:
Mapping of the dominant negative raf response region. A, dominant negative raf-induced stimulation requires the -1.7 to -1.3
kb upstream region of the 5`-UTR. HSCs were cotransfected with colCAT
reporter plasmids as described in Fig. 2. The
pdel1.3-0.4/1.6 plasmid contained the -0.4/1.6 plasmid
ligated to the -1.7 to -1.3 kb region of the 5`-UTR. The
-3.6siteTAE/1.6 plasmid contained the mutated TAE region
(depicted as a hatched oval) (mTAE(A)) in situ, as in Fig. 2. The relative amount of colCAT expression is displayed on
the right (
Figure 4:
Stellate cells bind the TAE. A,
gel retardation assays demonstrate selective binding to the TAE
oligonucleotide. Nuclear extracts (10 µg/lane) from HSCs were
incubated with radiolabeled double-stranded TAE and electrophoresed on
a native 6% polyacrylamide gel. Competition binding assays were
performed with varying amounts of unlabeled TAE or unlabeled mutated
TAE(A) or TAE(B), as indicated. The arrow on the left indicates the specific binding of TAE. B, activated
stellate cells contain a 60-kDa protein binding activity for the TAE.
Nuclear extracts (50 µg) from either culture-activated (C)
HSCs or quiescent HSCs (Q) obtained directly from normal rats
or rats given carbon tetrachloride 48 or 72 h prior to sacrifice were
resolved by SDS-PAGE, transblotted, and probed with radiolabeled
double-stranded TAE, using Southwestern blotting techniques. A single
60-kDa protein (p60) was detected in the lanes containing in vitro or in vivo activated stellate cell extracts but not in
the quiescent cell extract. Molecular size markers are shown on the left of the gel.
Since the TAE binding domain is
dissimilar to known transcription factor binding domains, p60 may
represent a novel transcription factor. Future studies will need to
characterize this protein further.
Volume 271,
Number 19,
Issue of May 10, 1996 pp. 11039-11042
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I(I) collagen gene expression in activated stellate cells. This
suggests an unappreciated branch point exists between Raf and MAPK. A
MAPK-stimulatory signal was mapped to the most proximal NF-1 and Sp-1
binding domains of the 5`-untranslated region of the collagen gene. A
Raf-inhibitory signal was mapped to a further upstream binding domain
involving a novel 60-kDa DNA-binding protein (p60). The cell-specific
expression and induction of p60 in stellate cells during the early
stages of hepatic fibrogenesis in vivo suggest a central role
for this pathway during liver injury and stellate cell activation.
Raf
MEK (
) MAPK series. Many putative MAPK nuclear
acceptor proteins have been suggested in the Egr, Ets, Fos, or AP-1
families(2, 3, 4, 5, 6, 7) .
The I(I) collagen gene contains AP-1 sites present in its 5`-UTR
and 1st intron. These cites could represent the downstream target of
the Ras
cascade(8, 9, 10, 11, 12, 13, 14, 15) . Raf and MAPK. A MAPK-stimulatory cascade is balanced with a Ras
Raf-inhibitory cascade. The two separate cascades mapped to two
distinct regions of the 5`-UTR, unrelated to AP-1 domains. The MAPK
cascade involved the ubiquitous Sp-1 and NF-1 transcription factors in
the proximal -100 bp domain. The Ras Raf cascade utilized
a more upstream -1680 bp domain. This latter domain involves a
novel 60-kDa DNA-binding protein, which is selectively produced by
activated stellate cells in culture and following activation in
vivo.
Cell Culture
HSC were isolated from
Sprague-Dawley male rats and subcultured by previously described
methods(16, 17) . Experimental manipulations were
performed with cells at passages 2-6.Transfection Studies
Stellate cells were
transfected using the LipofectAMINE reagent, and cell extract handling,
extraction, quantitation, and CAT measurements were performed as
described previously(22, 23) . Following transfection,
the cells were maintained in serum (10% fetal calf, 10% calf serum) for
48 h prior to CAT analysis. The plasmids used for transfection included
the intcolCAT plasmids (-3.6/1.6 or -1.7/1.6), which
contain either -3.6 or -1.7 kb of the 5`-UTR of the rat
I(I) collagen linked to the 1st exon and 1.6 kb of the 1st intron
and the CAT reporter gene(9) . The -1.3/1.6 intcolCAT
plasmid and the -0.4/1.6 intcolCAT plasmid were derivatives of
the -3.6/1.6 intcolCAT plasmid produced by digestion with NheI/TthIIII and NheI/MfeI
restriction endonucleases, respectively. Ends were then blunted and
ligated by T
DNA ligase. The pdel1.3-0.4/1.6 plasmid
was a derivative of the -1.7/1.6 plasmid, which was produced by
digestion with TthIIII/MfeI. This created a deletion
from -1284 bp to -392 bp, and then the ends were blunted
and ligated with T DNA ligase. The -3.6siteTAE/1.6
plasmid was created by overlap extension and contained the mutated
TGF
activation element (TAE(A)) described below(24) . All
mutated plasmids were sequenced to confirm the positions of the
mutations. All transfections contained equivalent amounts of plasmid
DNA (1.5 µg) by using empty vector pMNC plasmid. Cotransfections
utilized either pMNC or CMV-driven dominant negative raf (301-1 plasmid), dominant negative ras(N-17),
dominant negative or wild type MAPK (pCMVp41(Ala-54/55)
, G3BCAT (Sp-1 CAT reporter), or
BCAT-1 (Sp-1 CAT ``control'' reporter)
plasmids(2, 3, 4, 5, 6, 7) .
Transfection efficacy was monitored by parallel transfection with a
-galactosidase plasmid, as described
previously(22, 23) . Each data point represents the
mean of two sets of six pooled transfections. Variation between sets
was <15%, unless otherwise shown. Results are representative of at
least three independent experiments.I(I) collagen
gene). The following were the sequences of the oligonucleotides
used.
stimulation of collagen promoter activity (25) . In addition, TAE protein binding was abolished when
mTAE(A) or mTAE(B) was used(25) .Gel Retardation Assays
Nuclear extracts (10
µg/lane) from HSCs were incubated with radiolabeled double-stranded
TAE oliogonucleotides (Life Technologies, Inc.) and electrophoresed on
a native gel, as described(25) . Competition binding assays
were performed with unlabeled double-stranded oligonucleotides (TAE or
mutated TAE).Southwestern Blotting
Nuclear extracts (50 µg)
were obtained from either culture-activated stellate cells (HSC) or
fresh quiescent HSCs from normal rats. These were compared to stellate
cell extracts from activated cells obtained from rats that had been
pretreated with carbon tetrachloride (CCl) (0.5 ml +
0.5 ml of mineral oil given intraperitoneally) 48 or 72 h prior to
sacrifice. The extracts were resolved by SDS-PAGE, transblotted to a
nitrocellulose membrane, and probed with the radiolabeled
double-stranded TAE described above, using standard Southwestern
blotting techniques(25) .
Dominant Negative raf Versus Dominant Negative MAPK:
Differential Regulation of Collagen Gene Expression
Stellate
cells cotransfected with a collagen reporter gene and either dominant
negative raf plasmid or dominant negative MAPK plasmid yielded divergent changes in gene expression (Fig. 1A). Dominant negative raf transfection
resulted in a 5-fold increase in reporter expression. Surprisingly,
when dominant negative MAPK was transfected, a 3-fold decrease
in reporter expression was found. Previous HSC studies used these same
dominant negative plasmids to demonstrate raf's and MAPK's respective roles in insulin growth factor and
1,25-dihydroxyvitamin D
nuclear signaling(23) . By
varying the amount of input DNA, the optimal dominant negative raf or MAPK plasmid concentration was determined (Fig. 1, B and C). Based on previous studies
with these same plasmids, the optimal plasmid concentrations should
result in sufficient amounts of the mutated kinase proteins to bind the
upstream cascade proteins (either Ras or MEK,
respectively)(2, 3, 4, 5, 6, 7) .
However, the precise mechanism associated with the dominant negative
plasmid effect is unclear. The dominant negative MAPK suppression of reporter expression was observed in the absence (Fig. 1C) or presence of dominant negative raf (Fig. 1D). This reduction in colCAT expression by
dominant negative MAPK (1 (base line) versus 0.35
(dominant negative MAPK) (CAT units/mg of protein)) contrasts
markedly with the increase in colCAT expression (to 5.5 CAT units/mg of
protein) obtained by transfecting comparable amounts of the wild type
MAPK plasmid (data not shown). In other studies, dominant negative ras transfection caused a 3.3-fold increase in reporter
expression (data not shown), which mimics the dominant negative raf effect. These findings imply that the suppressive pathway is
likely to involve a Ras Raf
link(2, 3, 4, 5, 6, 7) .
Collectively, these results suggest that Raf activation leads to two
distinct effects on type I collagen promoter activity: (i) a
suppressive effect via a MAPK-independent pathway and (ii) a
stimulatory effect via a MAPK-dependent pathway. The latter effect is
also likely to involve Raf-parallel pathways, which utilize other
members of the enlarging MAPK kinase kinase
family(2, 7) . These studies further imply that there
is an additional branch point between Raf and MAPK. The Ras-Raf-MAPK
cascade could help regulate the expression of the major disease-related
collagen gene during fibrogenesis. Accumulated evidence suggests that
the diseased stellate cell initially responds to mitogenic stimuli (e.g. platelet-derived growth factor), which would be expected
to activate Ras Raf. The increase in stellate cell collagen gene
expression occurs at a later time point. The fibrogenic stimuli
responsible for this later increase are incompletely understood, but
transforming growth factor (TGF) is a likely
mediator(17, 18, 19, 20, 21) .
The TGF kinase cascade involves several unique kinases, but their
role in collagen gene expression is unknown(26, 27) .
Previous work suggests TGF may block Ras activation and therefore
decrease Raf activation (28) . During fibrogenesis, the
stellate cell may be initially exposed to activated Raf and MAPK (in a
platelet-derived growth factor-dominated stage). At the later
TGF-dominated stages, activated Raf levels may decrease. This
would be predicted to lead to an increase in collagen gene expression.
The relative duration of Raf versus MAPK activation or the
amount of nuclear activated MAPK could then determine the ultimate
extent of collagen gene activation. In view of the development of
selective MEK inhibitors, these observations may have therapeutic
importance(29) .
and maintained in serum (10%
fetal calf/10% calf serum)-containing media for 48 h prior to CAT
analysis. B, varying concentrations of dominant negative raf plasmid stimulate intcolCAT expression. HSCs were
cotransfected with increasing concentrations of dominant negative raf plasmid and intcolCAT. All transfections contained
equivalent amounts (1.5 µg) of plasmid DNA by using empty vector
pMNC plasmid. C, varying concentrations of dominant negative MAPK plasmid suppress intcolCAT expression. HSCs were
cotransfected with increasing concentrations of dominant negative MAPK plasmid and intcolCAT. All transfections contained
equivalent amounts (1.5 µg) of plasmid DNA by using empty vector
pMNC plasmid. D, increasing concentrations of dominant
negative MAPK suppresses dominant negative raf effect. HSCs were cotransfected with varying concentrations of
dominant negative MAPK and a constant dominant negative raf concentration or dominant negative MAPK alone.
All transfections contained equivalent amounts of plasmid DNA, as
above. Data are expressed as -fold increase (absolute number above each
point) versus cotransfection with empty pMNC
plasmid.
, pMNC; 
, dn-MAPK), with the absolute
amount of colCAT expression in the presence of the pMNC plasmid given
an arbitrary value = 1 for each individual reporter. The parent
intcolCAT reporter contains -3.6 kb of 5`-UTR region upstream of
the 1st exon (depicted as a black box), which is serially
linked to 1.6 kb of the 1st intron, the SV40 splice acceptor (depicted
as a gray box), and then the translation start site and the
chloramphenicol acetyltransferase gene (depicted as a striped
rectangle). The deletions (del) made in the 1st intron
are shown as empty rectangles. The mutation of the NF-1 site
of footprint 1 (codon substitutions GG TT at codon -96 and
-97)) is depicted as a dotted oval in its respective
plasmid (-3.6(FP-1mut)/1.6). When this plasmid was used in
cotransfections with pMNC, the absolute amount of CAT/mg protein was
4-5-fold reduced versus the -3.6/1.6 intcolCAT
reporter. The -3.6siteTAE/1.6 plasmid contained the mutated TAE
region (depicted as a hatched oval) (mTAE(A)) in
situ.
Dominant Negative raf Versus Dominant Negative MAPK
Utilize Different DNA Response Elements
To identify the DNA
response elements which are sensitive to the dominant negative (dn)
MAPK versus dominant negative (dn) raf effects,
truncated reporter constructs were substituted for the parent plasmid.
The dn-MAPK-sensitive region of the collagen gene was found to
be independent of the 1st intron, which contains AP-1 binding sites (Fig. 2)(12, 13, 14) . In addition,
most of the 5`-UTR appears to be dispensable. Recent studies have
suggested that the basal promoter activity lies within the most
proximal 200 base pairs, which contain consensus Sp-1 binding sites
(within a region termed footprint 2) and consensus NF-1 binding sites
(within a region termed footprint 1)(8, 30) .
Site-directed mutagenesis of the NF-1 site in footprint 1 abolished the
response to dn-MAPK (Fig. 2). In addition,
cotransfection experiments found that dn-MAPK caused a 50%
reduction in a Sp-1-driven CAT reporter versus no effect on a
control Sp-1 reporter (data not shown). Therefore, the MAPK-sensitive
region appears to involve both NF-1 and Sp-1 sites. Future studies will
be needed to determine if this is a direct effect on the
phosphorylation state of these transcription factors or an indirect
effect involving other MAPK-sensitive nuclear factors. stimulation of the type I collagen promoter(25) . This
effect was attributed to a novel 30-kDa protein identified by
Southwestern blotting(25) . Since preliminary studies suggested
that the TAE region was responsible for most of the DNA binding
activity of the -1.7 to -1.3 kb region in HSCs, further
studies concentrated on the TAE region. Site-directed mutagenesis of
the TAE region markedly reduced the dn-raf effect (see
-3.6siteTAE/1.6 reporter, Fig. 3A). In contrast,
the dn-MAPK effect was preserved when the same TAE mutated
reporter (see -3.6siteTAE/1.6 reporter, Fig. 2) was used.
To confirm that TAE binding was required in vivo for the
dn-raf effect, HSCs were cotransfected with the
pdel1.3-0.4/1.6 reporter and double-stranded TAE in a competition
assay (Fig. 3B). When 1 or 2 µg of TAE was used,
the dn-raf effect was abolished. A lesser blocking effect was
seen with 0.5 µg of TAE (data not shown). To control for
nonspecific binding, two mutants of TAE were used. These mutants (A and
B) contain codon substitution in the wild type TAE that eliminate
TAE-DNA binding in vitro (see below). When either TAEmut ((A)
or (B)) was substituted for wild type TAE, the dn-raf stimulatory effect on colCAT expression was preserved. TAEmut(A)
partially suppressed the dn-raf effect but it was much less
effective than wild type TAE. This suggests that TAEmut(A) retains some
binding activity in vivo that is eliminated in TAEmut(B). The
more stringent conditions of the in vitro gel retardation
assay (see below), however, suggest that the majority of TAE binding is
lost in TAEmut(A). These results collectively demonstrate that TAE
binding is required for dn-raf stimulation of colCAT
expression, and this involves the -1628 to -1615 bp domain.
, pMNC; , dn-raf). B,
dominant negative raf-induced stimulation is blocked by excess
TAE. HSCs were cotransfected with dominant negative raf and
the pdel1.3-0.4/1.6 plasmid ± 1 or 2 µg of
double-stranded TAE and maintained in serum-containing media for 48 h
prior to CAT analysis, as described
previously(22, 23) . Data are expressed (mean ±
standard deviation; n = 3) in terms of the amount of
dominant negative raf stimulation. An arbitrary value =
1.0 with transfection with wild type TAE is equivalent to no effect
with the dominant negative raf plasmid. Parallel
cotransfections utilized 1 or 2 µg of mutated TAE(A) or TAE(B).
Similar results were obtained when the -3.6/1.6 plasmid was used
(data not shown). , TAE; , mTAE(A); &cjs2117;,
mTAE(B).
TAE Binding Is Selective for in Vitro and in Vivo
Activated Stellate Cells
TAE characterization studies revealed
that HSCs contain a single retarded band when nuclear extracts are
incubated with radiolabeled TAE in a gel retardation assay (Fig. 4A). Binding specificity was confirmed by
competition binding assays with increasing concentrations of unlabeled
TAE but not by similar amounts of unlabeled mutated TAEs (mTAE(A) or
mTAE(B)). Southwestern blotting demonstrated a single 60-kDa binding
activity (p60) in cultured HSCs (Fig. 4B). This binding
activity is likely to have relevance during stellate cell activation
and fibrogenesis in vivo because an identical binding activity
was found in nuclear extracts from stellate cells activated in vivo after a single injection of CCl. Freshly isolated
stellate cells have very low levels of collagen gene expression and
lack this DNA binding activity (Fig. 4B)(8, 20) . CCl treatment increases stellate cell proliferation and collagen gene
expression during the immediate 48-72 h post-exposure and induces
fibrosis/cirrhosis after 6-12 weeks of chronic
exposure(20, 21) .
),
transforming growth factor ; dn, dominant negative; NF-1, nuclear
factor-1; FP-1, footprint 1; TAE, TGF activation element; CAT,
chloramphenicol acetyltransferase; PAGE, polyacrylamide gel
electrophoresis; bp, base pair(s); kb, kilobase pair(s).
We thank J. Mullen and J. Vande Vusse for technical
assistance, and U. Rapp, R. Davis, J. Leiden, V. Sukhatme, D. Rowe, D.
Breault, and R. Tjian for plasmids used in transfection.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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