(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
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.
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 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) .
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
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.
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 I(I) collagen
gene). The following were the sequences of the oligonucleotides
used.
Similar wild type and mutated TAEs have been shown previously to
selectively disrupt TGF stimulation of collagen promoter activity (25) . In addition, TAE protein binding was abolished when
mTAE(A) or mTAE(B) was used(25) .
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) 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.
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 (, 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.
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 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.
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 (, 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).
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.