From the
The TSK mouse, a model of fibrosis, displays exaggerated
connective tissue accumulation in skin and visceral organs including
the heart. To study the mechanisms of myocardial fibrosis in TSK mice,
we established several strains of TSK mice myocardial fibroblasts in
culture and examined the regulation of collagen gene expression in
these cells. These strains displayed increased collagen gene expression
in comparison with myocardial fibroblasts established from normal mice.
On an average, the TSK myocardial fibroblast cultures showed a 4-fold
increase in collagen synthesis and 4.4- and 3.6-fold increases,
respectively, in
These
observations indicate that a strong negative regulatory sequence
contained within -0.675 to -0.804 kilobase of the
A prominent feature of fibrosis is the accumulation of excessive
extracellular matrix components, especially types I and III collagens.
The TSK mice mutation, which is characterized by excessive collagen
accumulation in tissues such as skin and heart, is a prototype for
understanding the molecular pathogenesis of fibrosis (Green et
al., 1976; Jimenez et al., 1986; and Osborn et
al., 1987). TSK mice skin organ cultures as well as TSK mice
dermal fibroblasts display higher collagen synthesis, in comparison
with normal (Jimenez et al., 1984, 1986). The TSK mouse
myocardium shows enhanced collagen accumulation within the interstitium
(Osborn et al., 1987), displays thicker collagen type I and
collagen type III fibers and contains increased levels of
Overall, our observations
imply that decreased binding of AP-1 transcription factor to a negative
regulatory sequence between -0.675 and -0.804
kb
For Northern hybridization
analysis of RNA, samples of total RNA were electrophoresed on 8%
agarose, 2.2
M formaldehyde gels, transferred from the gels to
nitrocellulose filters, and the filters were prehybridized as described
previously (Jimenez et al., 1986). The filters were hybridized
at 45 °C overnight with [
Dot blot analysis was performed as
described by Sambrook et al. (1989) and Davis et al. (1986). Serial dilutions of RNA prepared from each cell strain
were made in loading buffer composed of 10
For mobility shift assays, three oligonucleotides spanning the
mapped cis-acting sequence were synthesized. The
oligonucleotides were end-labeled by incubating 100 ng of each of the
oligonucleotides, with [
For supershift assays, 10 µg of nuclear proteins
were incubated with c- jun/AP-1 antibody (Santa Cruz
Biotechnology Inc.) for 5 h at 4 °C and then subjected to DNA
binding reaction as described above.
The TSK mouse is an animal model for scleroderma, and it can
be considered a model for myocardial fibrosis. The present study was
undertaken to delineate the mechanisms responsible for the increased
collagen deposition and collagen gene expression in the TSK mouse
myocardium. Examination of the cellular origin of the myocardial
collagen network showed that, whereas cardiomyocytes participate in the
synthesis of type IV collagen, fibroblasts are the cellular origin of
collagen types I and III (Eghbali et al., 1988). Since
fibroblasts are responsible for myocardial collagen synthesis, normal
and TSK mice cultures of myocardial fibroblasts were established to
examine the mechanisms responsible for excessive collagen accumulation
in the TSK myocardium.
All TSK mice myocardial fibroblast strains
established showed increased synthesis of collagen, in comparison with
normal (). The observed increase in collagen biosynthesis
by TSK mice myocardial fibroblasts was substantially higher than the
19% increase in hydroxyproline content seen in 1-year-old TSK mouse
hearts, in comparison with normal (Osborn et al., 1987) and
higher than the 2-fold increase in [
Increased collagen biosynthesis in TSK fibroblasts was
associated with coordinate increases in mRNA steady state levels and
transcription rates of
Comparison of
the transcriptional activity of various
Since AP-1 is not a single protein but a complex whose
components are products of the fos and jun gene
families, the specific components involved in the altered collagen
expression in TSK fibroblasts need to be delineated. Each of the AP-1
components have been shown to have different functions. Furthermore,
AP-1 has been reported to act as a transactivator (Lee et al.,
1987; Angel et al., 1987) as well as a transrepressor (Franza
et al., 1988; Takimoto et al., 1989; Lian et
al., 1991) of various genes. c-Jun and Jun D play opposing roles
in growth regulation (Pfarr et al., 1994). Regarding collagen
genes it has been shown that an AP-1 site in the first intron of the
human
The results
reported here suggest that AP-1 is a transcriptional inhibitor of the
expression of the
Total RNA from normal and TSK
myocardial fibroblast cultures was examined by dot blot hybridizations
as described under ``Materials and Methods.''
Semiquantitative densitometric analysis of the blots following
standardization with GAPDH mRNA levels in each RNA preparation was
performed. The results are the averages of duplicate experiments and
are expressed as a TSK/normal ratio.
The autoradiograms of CAT assays
corresponding to each strain were analyzed by densitometry and the
areas corresponding to acetylated chloramphenicol were quantitated in
arbitrary densitometric units (CAT activity). The CAT activity driven
by each
1(I) and
1(III) collagen mRNA steady state
levels. The increased
1(I) and
1(III) collagen mRNA levels
were mainly due to increased transcription rates (3.4- and 3.8-fold
higher, respectively) of the respective genes. Furthermore, we showed
that the up-regulation of
1(I) procollagen gene transcription in
TSK mice myocardial fibroblasts was due to the lack of the strong
inhibitory influence of a regulatory sequence contained in the promoter
region encompassing nucleotides -675 to -804. Nuclear
extracts from TSK mice myocardial fibroblasts showed lower DNA binding
activity to oligonucleotides spanning the mapped regulatory sequence as
well as to a consensus AP-1 sequence, but not to a consensus SP-1
sequence, and supershift experiments with an AP-1 antibody confirmed
the interaction of these oligonucleotides with AP-1 protein.
1(I)
procollagen promoter binds AP-1 transcription factor and mediates
inhibition of gene transcription in normal murine myocardial
fibroblasts. The TSK mice myocardial fibroblasts lack this inhibitory
control, due to lower available amounts and/or decreased binding
activity to this inhibitory sequence, and hence display increased
1(I) procollagen gene expression.
2(I),
1(III), and
2(IV) procollagen mRNAs (Chapman and Eghbali,
1990). Higher collagen biosynthetic activity, collagen type VI content,
and
2(VI) collagen mRNA levels have also been demonstrated in
organ cultures, tissue, and cultured fibroblasts of TSK mice hearts in
comparison with normal (Bashey et al., 1993). Here, we
examined the expression of collagen types I and III in normal and TSK
mice myocardial fibroblasts and determined the cis-acting
regulatory elements governing transcriptional activity of an
1(I)
procollagen promoter in normal and TSK mice myocardial fibroblasts.
Furthermore, we examined the alterations in trans-acting
factors responsible for the up-regulation of type I collagen expression
in the TSK mice myocardial fibroblasts.
(
)
of the
1(I) procollagen promoter
resulted in increased transcriptional activity of
1(I) procollagen
gene which in turn led to enhanced procollagen mRNA levels and collagen
biosynthesis in TSK mice myocardial fibroblast strains, in comparison
with normal.
Establishment of Normal and TSK Mice Myocardial
Fibroblast Cultures
Hearts from six to eight heterozygous TSK
(Tsk/+) mice (about 6 months old) and their normal littermates
(pa/pa) were used for the establishment of each myocardial fibroblast
strain. Four strains of myocardial fibroblasts were isolated, cultured,
and passaged as described previously by Bashey et al. (1992).
Briefly, minced myocardial tissue pieces were incubated in Hanks'
buffer containing trypsin (0.1 µg/µl) and collagenase (50
units/ml) for 10 consecutive incubations of 10 min each at 37 °C.
Cells in the supernatants from each of the digestions were pelleted,
resuspended in culture media, and seeded on 100-mm tissue culture
dishes for 3 h, after which the incubation media containing nonadherent
cells was discarded and the plastic-attached cells were cultured. When
the cultures reached confluence, the cells were dissociated with
trypsin and subcultured as described previously (Bashey et
al., 1992).
Collagen Biosynthesis
Protein synthesis was
determined by measuring total [C]proline
incorporation in normal and TSK mice myocardial fibroblasts as
described by Jimenez et al. (1986). Biosynthesis of
[
C]collagenase-sensitive proteins was measured
by collagenase digestion of the
C-labeled proteins exactly
as described by Diegelmann and Peterkofsky (1972) and Peterkofsky and
Diegelmann (1971). The [
C]proline incorporation
and [
C]collagenase-sensitive proteins in media
and cells were normalized for the DNA content of the cultures which was
determined by DNA assays of aliquots of undialyzed cells as described
previously by Labarca and Paigen (1980). To characterize the newly
synthesized
C-labeled proteins, equal volume aliquots of
media and cells were electrophoresed on 7% SDS-PAGE under reducing
conditions as described by Bashey et al. (1992).
RNA Analysis
Confluent normal and TSK mice
myocardial fibroblasts cultures in T-175 flasks were incubated with
ascorbic acid (50 µg/ml media) for 24 h, and total RNA was
extracted from harvested cells employing the guanidine/cesium chloride
method (Sambrook et al., 1989).
-
P]CTP-labeled
murine
1(I) procollagen cDNA (French et al., 1985),
murine
1(III) procollagen cDNA (Liau et al., 1985) and
rat GAPDH cDNA (Marty et al., 1985). The filters were washed
under conditions of high stringency to 0.1
SSC, 0.1% SDS at 65
°C and exposed to x-ray films overnight. The films were developed
and scanned by densitometry.
SSC, to give final
RNA concentrations from 3 to 0.7 µg or from 4 to 0.5 µg. The
serial concentrations of RNA were dot-blotted and immobilized on
nitrocellulose filters. The filters were prehybridized and hybridized
with
1(I) procollagen,
1(III) procollagen, and GAPDH cDNAs as
described above. The filters were washed under conditions of high
stringency and exposed to x-ray films which were developed and scanned
by densitometry. To correct for differences in RNA loading and
transfer, the arbitrary densitometry units obtained from the
hybridizations with
1(I) procollagen or
1(III) procollagen
cDNAs were corrected for the arbitrary densitometric units obtained
from hybridizations with GAPDH cDNA.
Analysis of Transcriptional Rates
The
transcriptional rates of 1(I) procollagen and
1(III)
procollagen genes in normal and TSK mice fibroblasts were determined by
nuclear run-on assays (Sariban et al., 1988). Equal numbers of
normal and TSK mice myocardial fibroblasts were homogenized in
homogenization buffer (0.3
M sucrose, 10 m
M NaCl, 5
m
M MgCl
, 0.5 m
M dithiothreitol, 10
m
M Tris, pH 7.5, and 1% Triton X-100) and centrifuged at 2500
rpm at 4 °C for 5 min to pellet the nuclei. The pellets were then
resuspended in nuclei storage buffer (40% glycerol, 50 m
M
Tris, pH 8.0, 5 m
M MgCl
, and 0.1 m
M
EDTA). The isolated nuclei were incubated with transcription mixture
(90 m
M KCl, 3 m
M MgCl
, 400
µ
M ATP, GTP, and UTP, 2 m
M dithiothreitol, 1
unit/µl RNasin, and 4.1 µ
M
[
-
P]CTP) for 25 min at 25 °C. After
proteinase K and DNase digestions the RNA was extracted with
phenol/chloroform, precipitated, dissolved in hybridization solution,
and the radioactivity of the samples determined by scintillation
counting. Aliquots containing equal amounts of labeled RNA from normal
and TSK mice fibroblasts were hybridized to
1(I)procollagen,
1(III) procollagen, and GAPDH cDNAs immobilized on nitrocellulose
filters. The filters containing the bound cDNAs were washed, exposed to
x-ray films, and then counted in a liquid scintillation counter.
Analysis of Regulatory cis-Acting Elements
To
determine whether there were differences in the transcriptional
activity driven by various regulatory cis-acting elements in
the 1(I) procollagen gene between normal and TSK mice myocardial
fibroblasts, a series of human
1(I) procollagen promoter
constructs (5` end points from -5.3 kb to -0.08 kb) ligated
to the CAT reporter gene prepared as described previously (Jimenez
et al., 1994) were transiently transfected into both types of
cells. Plasmids were transfected into normal and TSK mice myocardial
fibroblast strains by calcium phosphate/DNA coprecipitation, followed
by glycerol shock for 1 min as described by Graham and Van Der Eb
(1973). Forty-eight h after transfection, cell extracts containing
equal amounts of protein were assayed for CAT activity by thin layer
chromatography (TLC). Autoradiographs of TLC plates were scanned, and
radioactive areas corresponding to acetylated and unacetylated
chloramphenicol in the thin layer TLC plates were cut and counted in a
liquid scintillation counter. The quantitative values obtained by
scanning and scintillation counting were similar. To monitor for
transfection efficiency, cells were cotransfected with pSV2AP, a
plasmid containing a full-length alkaline phosphatase cDNA fused to the
SV40 promoter and enhancer (Yoon et al., 1988), and alkaline
phosphatase activity in cell extracts was measured
spectrophotometrically.
Analysis of trans-Acting Factors
Nuclear protein
extracts were prepared from normal and TSK mice myocardial fibroblasts
as described by Andrews and Faller (1991) and the protein content of
the nuclear extracts was measured as described by Bradford (1976).
-
P]ATP (50
µCi), 1
kinase buffer and T4 polynucleotide kinase (8
units) for 1 h at 37 °C. The end-labeled oligonucleotides were
separated from unincorporated [
-
P]ATP by
passing the mixture through a G-25 Sephadex column. For DNA binding
reactions, 10 µg of nuclear extracts from normal or TSK myocardial
fibroblasts were incubated with 1
binding buffer (10
m
M HEPES, pH 7.9, 60 m
M KCl, 0.1 m
M EDTA,
and 1 m
M dithiothreitol), 1 µg of poly(dI-dC), 1%
glycerol, and 0.5 ng of labeled oligonucleotide on ice for 30 min in a
total volume of 10 µl. Following incubation, the DNA-protein
complexes were separated from free probe by electrophoresis on 4%
polyacrylamide gels with 1
Tris/glycine as running buffer. The
gels were dried and exposed to x-ray films overnight. The
autoradiographs obtained were scanned in a laser densitometer and the
arbitrary densitometric units obtained were expressed as ratios of
normal/TSK.
Establishment of Normal and TSK Mice Myocardial
Fibroblast Strains
In order to study the mechanisms responsible
for the accumulation of collagen in myocardial fibrosis and to examine
the regulation of collagen gene expression in myocardial fibroblasts,
we successfully isolated and cultured fibroblastic cells from normal
and TSK mice myocardium. Four normal and four TSK mice myocardial
fibroblast strains were established and are referred to as strains 1,
2, 3, and 4. Morphologically, fibroblasts derived from normal mice
myocardium had characteristic elongated spindle shapes, multiple
nucleoli, and agranular cytoplasm. Myocardial fibroblast cultures from
TSK mice showed appreciable differences in morphology with most of the
cells consistently displaying a star-shaped or rounded cell shape (not
shown). The cultures established could be passaged serially for at
least 12 passages without apparent changes in their morphology or in
their patterns of growth and proliferation.
Collagen Biosynthesis by Normal and TSK Mice Myocardial
Fibroblast Strains
represents total
[C]proline incorporation and
[
C]collagenase-sensitive proteins in media and
cell layers of various normal and TSK mice myocardial fibroblast
strains. In one TSK myocardial fibroblast strain the average total
[
C]proline incorporation and
[
C]collagenase-sensitive proteins, respectively,
were 3.8- and 1.5-fold higher in passage-2 cultures and 3.7- and 5-fold
higher in passage-8 cultures, in comparison with normal (not shown).
The average total [
C]proline incorporation and
[
C]collagenase-sensitive proteins, respectively,
were 2.5- and 2.3-fold higher in a second TSK mice myocardial
fibroblast strain, 2.2- and 3.4-fold higher in a third TSK mice
myocardial fibroblast strain, and 2.7- and 5.6-fold higher in a fourth
TSK mice myocardial fibroblast strain, in comparison with normal
(). In all four TSK mice myocardial fibroblast strains,
there were much greater increases in the
[
C]collagenase-sensitive proteins in the cell
layers than in the media (). On an average, the increases
in [
C]proline-labeled proteins in media and cell
layers, respectively, were 2- and 3.4-fold higher in TSK mice
myocardial fibroblast strains, whereas the increases in
[
C]collagenase-sensitive proteins in the TSK
mice strains were 1.8-fold higher in media and 10-fold higher in cell
layers, in comparison with normal. Furthermore, these differences in
collagen biosynthesis were also reflected in SDS-PAGE autoradiograms
which showed an increase in collagen in the media (not shown) but a
much greater increase in cell layer-associated collagen in TSK
fibroblast cultures, relative to normal (Fig. 1).
Figure 1:
SDS-PAGE analysis of
C-labeled proteins in cell layers of normal ( N)
and TSK mice myocardial fibroblast strains. Equal volume aliquots of
labeled cell layers were electrophoresed on 7% SDS-PAGE under reducing
conditions.
Steady State Levels of
The mRNA levels of types I and III
procollagens were analyzed to determine whether the increased collagen
biosynthesis in TSK mice myocardial fibroblast cultures was due to
increased collagen mRNA steady state levels. In comparison with normal,
the TSK mice myocardial fibroblast strains showed increased 1(I) Procollagen and
1(III) Procollagen mRNA in Normal and TSK Mice Myocardial
Fibroblast Strains
1(I)
procollagen and
1(III) procollagen mRNA steady state levels as
analyzed by Northern and dot blot hybridizations. As expected, two
1(I) procollagen transcripts corresponding to 5.7 and 4.7 kb were
seen in Northern blots hybridized with
1(I) procollagen cDNA,
whereas only 5.4-kb transcripts were found in the Northern blots
hybridized with
1(III) procollagen cDNA (Fig. 2). The mRNA
steady state levels of
1(I) procollagen and
1(III)
procollagen were quantitatively analyzed relative to GAPDH, a
non-collagenous constitutive protein, by scanning autoradiograms of
Northern and dot blot hybridizations and standardizing procollagen mRNA
levels with GAPDH mRNA levels in the respective RNA preparations
(). All the TSK myocardial fibroblast strains showed
greater than 2-fold increases in types I and III procollagen mRNAs and
in one cell strain (strain 4) the increases were 9.0- and 4.5-fold,
respectively. The increased collagen mRNA steady state levels
corresponded with the increased collagen biosynthetic activity of these
TSK myocardial fibroblast strains, suggesting altered regulation of
collagen gene expression in TSK mice myocardial fibroblast strains at a
pretranslational level.
Figure 2:
Northern hybridization of total RNA from a
normal and a TSK mice myocardial fibroblast strain. Total RNA was
electrophoresed on 8% formaldehyde-agarose gels, transferred to
nitrocellulose filters, and the filters were prehybridized and
hybridized with labeled cDNAs, washed, and exposed to x-ray film.
A, hybridization with 1(I) procollagen and GAPDH cDNAs;
B, hybridization with
1(III) procollagen
cDNA.
Transcription Rates of
Nuclear run-on assays using cDNAs for types I and III
procollagens indicated that the increased mRNA levels for these
proteins in cultured TSK mice myocardial fibroblasts were due to
increased transcription of the respective genes (Fig. 3). In
comparison with normal, the transcription rate of the 1(I) and
1(III)
Procollagens Genes in Normal and TSK Mice Myocardial Fibroblast
Strains
1(I)
procollagen gene in TSK mice myocardial fibroblast strains 2, 3, and 4,
following standardization for the transcription rate of the GAPDH gene
was, respectively, 2.3-, 3.4-, and 4.4-fold higher. With the exception
of strain 4, the observed increases in transcription rates of the
1(I) procollagen gene in these TSK mice myocardial fibroblast
strains paralleled the increases in steady state
1(I) procollagen
mRNA levels in the respective strains. Hybridization of labeled
transcripts with
1(III) procollagen cDNA showed that the
transcription rates
1(III) gene were 3.4- and 6.4-fold higher in
TSK mice myocardial fibroblast strains 2 and 4, respectively, in
comparison with normal. These values also largely corresponded with the
observed increase in
1(III) procollagen mRNA levels in these
strains. The transcription rate of GAPDH gene was fairly similar in
normal and TSK mice myocardial fibroblast strains. Control
hybridizations of the labeled transcripts from normal and TSK mice
fibroblasts to filters containing the plasmid pBR322 showed only very
faint radioactivity indicating the specificity of the
1(I)
procollagen,
1(III) procollagen, and GAPDH cDNAs for their
respective transcripts. The parallel increases in transcription rates
of
1(I) procollagen and
1(III) procollagen genes in TSK mice
myocardial fibroblast strains imply the involvement of common
transcriptional regulatory mechanisms. Furthermore, since the increase
in collagen biosynthesis in TSK mice myocardial fibroblast strains
correlated with the increases in collagen mRNA steady state levels,
which in turn correlated with increased transcription rates of
procollagen genes, it can be concluded that the up-regulation of
procollagen gene expression in TSK mice myocardial fibroblast strains
is mediated transcriptionally.
Figure 3:
Transcriptional activities of 1(I)
procollagen,
1(III) procollagen and GAPDH genes in normal and TSK
mice myocardial fibroblast strains. Initiated transcripts in nuclei
isolated from normal and TSK mice myocardial fibroblast strains were
elongated in the presence of [
P]UTP. The
radiolabeled transcripts were extracted from nuclei and hybridized to
1(I) procollagen,
1(III) procollagen, and GAPDH cDNAs
immobilized on nitrocellulose filters. The filters were washed and
exposed to x-ray films overnight.
Functional Analysis of Regulatory cis-Acting Elements of
the Human
A series of human 1(I) Procollagen Promoter in Normal and TSK Mice
Myocardial Fibroblast Strains
1(I)
procollagen promoter-CAT constructs (5` end points at -5.3,
-2.3, -0.804, -0.675, -0.463, -0.369,
-0.174, and -0.084 kb) were transiently transfected into
TSK and normal mice myocardial fibroblast cultures to map the
regulatory sequences responsible for the up-regulation of promoter
activity in TSK fibroblast cultures (Fig. 4). The transfection
efficiency was monitored by cotransfecting each of the procollagen
promoter deletion constructs with the pSV2AP plasmid followed by the
measurement of alkaline phosphatase activity in whole cell extracts. No
significant differences were found in alkaline phosphatase activity
between normal and TSK myocardial fibroblast strains, indicating that
the transfection efficiency was similar in both types of cells (results
not shown).
Figure 4:
CAT activity of a series of 1(I)
procollagen promoter-CAT constructs with 5` end points from -5.3
to -0.08 kb, transfected into normal ( N) and TSK
( T) mice myocardial fibroblast strains.
1(I) procollagen
promoter deletion constructs and pSV2CAT plasmid, respectively, were
transfected into normal and TSK mice myocardial fibroblasts by calcium
phosphate precipitation, and cell extracts were assayed for CAT
activity. The acetylated ( A) and nonacetylated ( C)
chloramphenicol were separated by thin layer chromatography and exposed
to x-ray film overnight at room temperature. A, strain 3;
B, strain 4.
The results of these studies with strains 3 and 4 are
shown in Table III and are illustrated in Fig. 4. Maximal
differences in CAT activity between normal and TSK strains were
consistently seen with the sequence from -0.804 to -0.675
kb, thus mapping this region as the principal region involved in the
differential regulation of promoter activity between normal and TSK
strains. Quantitative analysis of these results showed that in
comparison with normal, the CAT activity of the -0.804 kb
1(I) procollagen promoter-CAT construct was 4.6-fold higher in TSK
mice fibroblast strain 3 and 6.5-fold higher in TSK mice fibroblast
strain 4. Since inclusion of the promoter sequence from -0.804 to
-0.675 kb caused a reduction in CAT expression in normal
fibroblast strains relative to the activity driven by shorter
1(I)
procollagen promoter constructs, it can be inferred that this region is
inhibitory. In contrast, inclusion of this promoter sequence (from
-0.804 to -0.675 kb) did not alter CAT expression in TSK
fibroblast strains, indicating that the inhibitory control exerted by
this sequence in normal cells is absent in TSK strains. The slight
increase in CAT expression (
2-fold) with the -0.675- to
-0.463-kb promoter construct in TSK strains suggests that the
involved regulatory region may extend slightly further downstream from
the -0.675-kb end point. The CAT activity driven by the shorter
1(I) procollagen promoter-CAT constructs, with 5` end points at
-0.463, -0.369, -0.174, and -0.084 kb, was
similar in normal and TSK myocardial fibroblasts. Overall, the markedly
higher CAT expression in the presence of the sequence from -0.804
to -0.675 kb
1(I) procollagen promoter in TSK mice
myocardial fibroblast strains, in comparison with normal, provides
strong evidence that the major regulatory elements responsible for the
up-regulation of
1(I) procollagen gene expression in TSK mice
myocardial fibroblast strains are located in this region.
Interaction of trans-Acting Factors in Nuclear Extracts
from Normal and TSK Mice Myocardial Fibroblast Strains with the Mapped
cis-Acting Regulatory Sequence
To examine the interaction of
trans-acting factors with the regulatory region in the 1(I)
procollagen promoter that showed maximal differences between normal and
TSK fibroblast strains, DNA binding proteins present in the nuclei of
normal or TSK myocardial fibroblasts that recognize sequences within
this region were examined. For this purpose, end-labeled
oligonucleotides 1, 2, and 3, which correspond, respectively, to
sequences -0.804 to -0.763 kb, -0.762 to -0.718
kb, and -0.717 to -675 kb of the
1(I) procollagen gene
(Fig. 5) were incubated with equal amounts of nuclear proteins
from normal and TSK mice myocardial fibroblasts. As shown in
Fig. 6
, this interaction resulted in the formation of specific
DNA-protein complexes. The DNA-protein complexes formed with nuclear
extracts from normal and TSK mice fibroblasts and labeled
oligonucleotides 1 (Fig. 6 A), 2
(Fig. 6 B), and 3 (Fig. 6 C), respectively,
displayed qualitatively similar electrophoretic mobilities. The labeled
oligonucleotide-protein complexes formed were specific because their
formation was not abolished by excess salmon sperm DNA, whereas it was
abolished by excess amounts of the corresponding unlabeled
oligonucleotide. Mobility shift assays showed that nuclear extracts
from all TSK mice myocardial fibroblast strains contained much lower
binding activity for each of the three oligonucleotides than nuclear
extracts from normal mice fibroblasts (). On an average,
in comparison with normal, the binding activities for oligonucleotides
1, 2, and 3 were, respectively, 3-, 3.7-, and 3-fold lower in TSK mice
myocardial fibroblasts. These results suggest that the nuclear extracts
from TSK mice fibroblasts contain lower binding activity for each of
the three oligonucleotides.
Figure 5:
Oligonucleotides used in mobility shift
assays. A, sequences of three oligonucleotides (1, 2, and 3,
respectively) spanning the 1(I) procollagen promoter sequence from
-804 to -675 base pairs. The AP-1 binding sites in these
sequences are shown. B, homologies between the consensus AP-1
sequence and the AP-1 binding sites present within -804 to
-675 base pairs of the
1(I) procollagen
promoter.
Figure 6:
Mobility shift assays with nuclear
extracts from a normal and a TSK mice myocardial fibroblast strain and
end-labeled double-stranded oligonucleotides. The
oligonucleotide-protein complexes ( arrow or bracket)
formed with 10 µg each of nuclear extracts from a normal and a TSK
mice fibroblast strain incubated with 0.5 ng each of labeled
oligonucleotide are shown. A, oligonucleotide 1; B,
oligonucleotide 2; C, oligonucleotide 3. Competition
experiments ( Comp.) were performed with 100-fold molar excess
of unlabeled oligonucleotides 1, 2, 3, or AP-1.
Since all three oligonucleotides showed
lower DNA-protein complex formation with nuclear extracts from TSK mice
fibroblasts, in comparison with normal cells, and each contains
transcription factor AP-1 binding sites, it seemed likely that each of
the three oligonucleotides were bound by the same trans-acting
factor. To examine this possibility, the ability of the
oligonucleotides to compete with each other for DNA-protein complex
formation was studied (Fig. 6). Oligonucleotides 1 and 3 competed
efficiently with each other for protein binding. Whereas
oligonucleotide 2 only partially abolished the formation of
oligonucleotide 1- and 3-protein complexes, oligonucleotides 1 and 3
abolished the formation of oligonucleotide 2-protein complex. In
addition, the observation that oligonucleotide 1-, 2-, and 3-protein
complexes, respectively, migrated with similar electrophoretic mobility
suggests that the three oligonucleotides bind the same
trans-acting factor.
Role of AP-1 Binding Sites and Identity of the Involved
trans-Acting Factor
The -0.804 to -0.675-kb
1(I) procollagen promoter sequence contains 5 AP-1 binding sites
which are preserved in the synthesized oligonucleotides and are
homologous to the consensus AP-1 sequence (Fig. 5). Thus, the
role of the AP-1 binding sites contained within the oligonucleotides
was examined by competition experiments with excess unlabeled AP-1
consensus oligonucleotide (Fig. 6) and by using a consensus AP-1
oligonucleotide as a probe in mobility shift experiments as described
by Kahari et al. (1992). The consensus AP-1 oligonucleotide
abolished the formation of oligonucleotide 1-, 2-, and 3-protein
complexes (Fig. 6). Mobility shift experiments with the labeled
consensus AP-1 oligonucleotide showed the presence of DNA-protein
complexes with nuclear extracts from normal and TSK fibroblasts
( bracket in Fig. 7). Furthermore, it was found that markedly
lower amounts of DNA-protein complexes were formed when nuclear
extracts from TSK mice myocardial fibroblasts were examined, in
comparison with nuclear extracts from normal cells ( Fig. 7and
). These complexes were specific because they were
competed out by excess unlabeled AP-1 oligonucleotide, but they were
not affected by addition of salmon sperm DNA. Competition experiments
showed that formation of these complexes was largely abolished by
oligonucleotides 1, 2, or 3 (Fig. 7). As a control to determine
the specificity of the findings of lower DNA binding with the AP
consensus oligonucleotide in nuclear extracts from TSK fibroblasts
compared with normal cells, we examined DNA binding activity to the
consensus SP-1 sequence (Fig. 8). We found that nuclear extracts from
normal and TSK mice myocardial fibroblasts showed no differences in the
amounts of DNA-protein complexes formed with the consensus SP-1
sequence ( arrows in Fig. 8). These observations indicate
that the nuclear extracts from TSK mice myocardial fibroblast strains
have lower AP-1 sequence binding activity than normal fibroblasts.
Figure 7:
Mobility
shift assays with nuclear extracts from a normal and a TSK mice
myocardial fibroblast strain and end-labeled double-stranded
oligonucleotide AP-1. The oligonucleotide AP-1-protein complexes
( bracket) formed with 10 µg each of nuclear extracts from
a normal and a TSK mice fibroblast strain incubated with 0.5 ng each of
labeled oligonucleotide AP-1 are shown. The specificity of the
oligonucleotide AP-1-protein complex was determined by inclusion of
100-fold molar excess of salmon sperm DNA and unlabeled oligonucleotide
AP-1, respectively, in the binding reaction. Competition experiments
( Comp.) were also performed with 100 fold molar excess of
unlabeled oligonucleotides 1, 2, or 3.
Figure 8:
Mobility shift assays with nuclear
extracts from normal ( N) and TSK mice myocardial fibroblast
strains and end-labeled double-stranded oligonucleotide SP-1. The
oligonucleotide SP-1-protein complexes ( arrows) formed with 10
µg each of nuclear extracts from normal and TSK mice fibroblasts
incubated with 0.5 ng of labeled oligonucleotide SP-1 are shown: A, TSK
strain 2; B, TSK strain 3; C, TSK strain
4.
In order to analyze the involvement of AP-1 trans-acting
factor, supershift experiments with an AP-1 antibody were performed.
Incubation of nuclear extracts with a polyclonal AP-1 antibody prior to
the DNA-protein binding reaction resulted in supershifting of some of
the oligonucleotide protein complexes (Fig. 9). Thus, the
1(I) procollagen promoter sequence between -0.804 and
-0.675 kb binds AP-1 transcription factor. Collectively, since
TSK nuclear extracts showed lower binding activity to a consensus AP-1
sequence as well as to AP-1 binding oligonucleotides spanning the
mapped regulatory sequence, it can be inferred that altered interaction
of AP-1 factor with the AP-1 binding sites in this region of the
1(I) procollagen promoter is involved in the up-regulation of
expression of the
1(I) procollagen gene in the TSK fibroblasts.
Figure 9:
Mobility
shift assays of nuclear extracts from normal and TSK myocardial
fibroblasts with end-labeled double-stranded oligonucleotides and a
c- jun/AP-1 antibody. Nuclear extracts from normal and TSK mice
myocardial fibroblasts were incubated with AP-1 antibody (+) for 5
h at 4 °C prior to adding labeled oligonucleotides to the binding
reactions for mobility shift assays. A, normal; B,
TSK. Oligonucleotide 1 was employed in experiments shown in A,
and oligonucleotide 3 was employed in experiments shown in B.
Note supershifting of a large proportion of the oligonucleotides 1 and
3 in the presence of c- jun/AP-1
antibody.
C]proline
incorporation and collagen synthesis observed in myocardial organ
cultures of TSK mice, in comparison with normal (Bashey et
al., 1993). Some explanations for the differences between in
vivo and in vitro results include suppression of
fibroblast biosynthetic activity in vivo by factors secreted
by other cell types such as cardiac myocytes or due to the presence of
a heterogeneous group of fibroblasts in vivo and the selection
of fibroblasts with an abnormal phenotype during culture in
vitro. The marked increase in cell layer-associated
[
C]collagenase-sensitive proteins in all TSK
mice myocardial fibroblast cultures implies the formation of a well
organized pericellular matrix. These observations indicate that the TSK
mice myocardial fibroblast strains are different from TSK mice dermal
fibroblasts, which in comparison with normal, show increases in
C-labeled proteins and collagen content only in media
(Jimenez et al., 1986). Interestingly, the cell layers of TSK
mice myocardial fibroblast cultures contained mostly newly synthesized
procollagen (Fig. 1) which is in contrast to cultured fibroblast
cell layers from other tissues and species which mainly contain
processed collagenous polypeptides (Bashey et al., 1983,
1992). The above observations collectively suggest that the increased
collagen deposition in the TSK mouse heart is largely due to the
increased collagen biosynthetic activity of TSK mice myocardial
fibroblasts.
1(I) and
1(III) procollagen genes. The
average steady state levels of
1(I) and
1(III) procollagens
mRNAs were 4.4- and 3.6-fold higher, respectively, in TSK mice
myocardial fibroblast strains, in comparison with normal
(). The observed levels are much higher than the 41 and
63% increases, respectively, in
1(I) and
1(III) procollagen
mRNA levels in the TSK mouse myocardial tissue (Chapman and Eghbali,
1990) but similar to the 5-fold increase in
1(I) and
1(III)
procollagen mRNA levels in TSK mice dermal fibroblast cultures (Jimenez
et al., 1986). Nuclear run-on assays showed that the increased
1(I) and
1(III) procollagen mRNA levels in TSK mice
myocardial fibroblast strains were largely due to increased
transcription of the respective genes (Fig. 3).
1(I) procollagen promoter
deletion constructs (5` end points from -5.3 to -0.084 kb)
in normal and TSK mice myocardial fibroblasts indicated that the
sequence between -0.675 and -0.804 kb of the
1(I)
procollagen gene is strongly inhibitory in normal mice fibroblasts but
its function is altered in TSK mice myocardial fibroblasts, thus
resulting in increased expression of the gene in TSK fibroblasts (Fig.
4). Additional evidence supporting the inhibitory role of this region
of the gene was provided by the studies of Simkevich et al. (1992), who found that deletion of the human
1(I) procollagen
promoter sequence from -0.609 to -0.804 kb caused high
collagen expression in transient transfections of mesenchymal and non
mesenchymal cells (Simkevich et al., 1992). Furthermore,
nuclear extracts from TSK fibroblasts showed reduced binding activity
to oligonucleotides encompassing the inhibitory region (between
-0.675 and -0.804 kb) of the
1(I) procollagen
promoter, in comparison with normal (Fig. 6). Hence, it can be
concluded that the increased transcriptional activity of the
1(I)
procollagen promoter in TSK mice myocardial fibroblast strains is due
to the decreased available amounts and/or lower DNA binding activity of
a transcriptional inhibitor that normally interacts with the strong
inhibitory sequence between -0.675 and -0.804 kb of the
1(I) procollagen gene to modulate transcription. We also observed
that oligonucleotides containing AP-1 binding sites corresponding to
this region of the gene showed lower DNA-protein complex formation with
nuclear extracts from TSK mice myocardial fibroblast strains (Figs. 6
and 7), and supershift experiments with an AP-1 antibody demonstrated
that these oligonucleotides bind AP-1 protein (Fig. 9). Overall,
these observations imply that decreased binding of AP-1 transcription
factor to a strongly negative regulatory sequence between -0.675
and -0.804 kb of the
1(I) procollagen promoter may be
responsible for the increased transcriptional activity of
1(I)
procollagen gene which in turn resulted in the enhanced procollagen
mRNA levels and collagen biosynthesis in TSK mice myocardial fibroblast
strains.
1(I) procollagen gene acts either positively or negatively
in different collagen producing cell lines (Katai et al.,
1992). The results reported here provide evidence that the regulatory
sequence between -0.675 and -0.804 kb of the human
1(I) procollagen gene, which contains multiple AP-1 binding sites,
is inhibitory in normal mice myocardial fibroblasts and may mediate
transrepression by binding to AP-1 protein. These observations are
consistent with studies of the effects of thyroid hormone and phorbol
myristate acetate on myocardial fibroblasts (Eghbali et al.,
1991; Yao and Eghbali, 1992) which showed that thyroid hormone
down-regulates collagen synthesis, type I procollagen mRNA steady state
levels, and
1(I) procollagen promoter activity as well as induces
c- fos and c- jun protooncogenes in cultured myocardial
fibroblasts (Yao and Eghbali, 1992). Similarly, treatment of cardiac
fibroblasts with phorbol myristate acetate causes decreased collagen
synthesis and procollagen mRNA steady state levels, effects which are
associated with increased expression of protooncogenes c- fos and c- jun (Eghbali et al., 1991).
1(I) procollagen gene and that alterations in
this regulatory mechanism are associated with the increased production
of collagen in TSK mice myocardial fibroblasts. The link between the
TSK mutation and associated multiple connective tissue abnormalities in
the TSK mice is not yet known. However, it is unlikely that reduced
AP-1 factor binding is a direct consequence of the TSK mutation,
because given the pleiotropic effects of AP-1 factor, it would be
expected that alterations in AP-1 factor expression/binding would
result in a wider spectrum of abnormalities than those in connective
tissue seen in the TSK mouse. It is possible that the TSK mutation
causes either activation or silencing of a gene product that leads to a
cascade of events, one of which is inhibition of AP-1 expression or
binding activity. However, cloning and identification of the TSK gene
and characterization of its product(s), and identification of its
functional role will be required in order to understand the cause of
the observed collagen abnormalities in the TSK mouse.
Table:
Collagen biosynthesis by normal and TSK mice
myocardial fibroblast strains
Table:
Steady state levels
of 1(I) and
1(III) procollagen mRNA in normal and TSK mice
myocardial fibroblast strains
Table:
Quantitation of CAT activity of a series of
1(I) procollagen promoter-CAT constructs with 5` end points from
-5.3 to -0.084 kb, transfected into normal (N) and TSK mice
myocardial fibroblasts
1(I) procollagen construct was corrected for efficiency of
transfection as described under ``Materials and Methods,''
and the values obtained are shown. Comparison of expression of the
various
1(I) procollagen promoter-CAT constructs in normal and TSK
mice myocardial fibroblasts is represented as the ratio of TSK/normal.
ND, not done.
Table:
Comparison of DNA binding activities of
nuclear extracts from normal (N) and TSK mice myocardial fibroblast
strains 2, 3, and 4 for oligonucleotides 1, 2, 3, or AP-1
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.