From the Departments of We have previously shown that transforming growth
factor- The collagens comprise a superfamily of proteins that play a
critical role in the maintenance of extracellular matrix integrity. Type VII collagen is found primarily in the basement membrane zone of
specialized squamous epithelia, such as in the skin, various mucous
membranes, and the cornea of the eye (1, 2). It is the predominant, if
not the exclusive, component of anchoring fibrils, attachment
structures that play a critical role in ensuring stability to the
association of the epithelial basement membrane zone to the underlying
papillary dermis (3, 4). Synthesis of functional anchoring fibrils is
of critical importance in providing integrity to the cutaneous basement
membrane zone, and abnormalities in these adhesion structures
clinically manifest as dystrophic forms of epidermolysis bullosa, a
group of heritable bullous diseases characterized by cutaneous
fragility and the tendency to sub-basal lamina densa blister formation
(5). In fact, our laboratory recently demonstrated that mutations
within the COL7A1 gene are associated with different forms of
dystrophic epidermolysis bullosa (6, 7).
Analysis of the 5'-end sequences of the human COL7A1 gene has revealed
a promoter devoid of a canonical TATA or CAAT box (GenBank/EMBL accession no. L23982). Its expression requires the integrity of an
Sp1-binding site located between residues In this study, we have investigated the molecular mechanisms by which
TGF- Cell Cultures--
Human dermal fibroblast cultures, established
by explanting tissue specimens obtained from neonatal foreskins, were
utilized in passages 3-6. The cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
calf serum, 2 mM glutamine, and antibiotics (100 units/ml
penicillin, 50 µg/ml streptomycin-G, and 0.25 µg/ml
FungizoneTM). Human recombinant TGF- Plasmid Constructs--
To study the transcriptional regulation
of human type VII collagen gene (COL7A1) expression, transient
transfection experiments were performed with various COL7A1 promoter
5'-deletion fragments cloned into promoterless pBS0CAT vector (13), as
described previously (8).
Transient Cell Transfections and CAT Assays--
Transient cell
transfections of human dermal fibroblasts were performed with a calcium
phosphate/DNA co-precipitation procedure (14). Briefly, cultured cells
were transfected with 10 µg of plasmid DNA and 2 µg of the
pRSV- Electrophoresis Mobility Shift Assays--
Several fragments
spanning the region between nucleotides TGF- Dermatology and Cutaneous
Biology and
Biochemistry and Molecular Pharmacology,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(TGF-
) increases type VII collagen gene (COL7A1)
expression in human dermal fibroblasts in culture (Mauviel, A.,
Lapière, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight
into the molecular mechanisms underlying the up-regulation of COL7A1 by
this growth factor, we performed transient cell transfections with a
series of 5'-deletion promoter/chloramphenicol acetyltransferase
reporter gene constructs. We identified a 68-base pair region between
nucleotides
524 and
456, relative to the transcription start site,
as critical for TGF-
response. Using electrophoresis mobility shift
assays (EMSAs) with an oligonucleotide spanning the region from
524
to
444, we discovered that a TGF-
-specific protein-DNA complex was
formed as early as 11 min after TGF-
stimulation and persisted for
1 h after addition of the growth factor. Deletion analysis
of the TGF-
responsive region of the COL7A1 promoter by EMSA
identified segment
496/
444 as the minimal fragment capable of
binding the TGF-
-induced complex. Furthermore, two distinct
segments,
496/
490 and
453/
444, appeared to be necessary for
TGF-
-induced DNA binding activity, suggesting a bipartite element.
Supershift experiments with a pan-Smad antibody unambiguously
identified the TGF-
-induced complex as containing a Smad member.
This is the first direct identification of binding of endogenous Smad
proteins to regulatory sequences of a human gene.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
512 and
505, relative to
the transcription start site (8). Despite these structural features
usually associated with promoters of so-called "housekeeping"
genes, type VII collagen expression has been shown to be
transcriptionally regulated by several cytokines, including transforming growth factor-
(TGF-
)1 (9-11), and by
other biological response modifiers, such as ultraviolet irradiation
(12). Interestingly, we have previously shown that cytokine-mediated
regulation of COL7A1 gene expression is strikingly different from that
of type I collagen, as evidenced by synergistic activation of COL7A1
gene expression by TGF-
and tumor necrosis factor-
(11). Thus
far, however, little is known about the transcriptional mechanisms
underlying the above mentioned regulation of COL7A1 gene
expression.
up-regulates the activity of human COL7A1 in dermal fibroblasts. We report, for the first time, evidence for Smad-mediated, immediate-early activation of a human gene by TGF-
through direct interaction of a Smad-containing transcription complex with the TGF-
-responsive region of the COL7A1 promoter.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 was a
kind gift from Dr. David Olsen, Celtrix Co., Palo Alto, CA. It is
referred to as TGF-
throughout the text.
-galactosidase plasmid DNA to monitor the transfection
efficiencies (15). After glycerol shock, the cells were placed in
Dulbecco's modified Eagle's medium containing 1% fetal calf serum,
and TGF-
was added 3 h later. After 40 h of incubation,
the cells were rinsed once with phosphate-buffered saline, harvested by
scraping, and lysed in 200 µl of reporter lysis buffer (Promega,
Madison, WI). The
-galactosidase activities were measured according
to a standard protocol (15). Unless stated otherwise, aliquots
corresponding to identical
-galactosidase activity were used for
each CAT assay with [14C]chloramphenicol as substrate
(16), using thin layer chromatography. Following autoradiography, the
plates were cut and counted by liquid scintillation to quantify the
acetylated [14C]chloramphenicol.
524 and
444 of the COL7A1
promoter responsive to TGF-
were generated by polymerase chain
reaction amplification using the plasmid
722COL7A1 as template and
purified by electroelution after electrophoresis in a 2% agarose gel.
Each oligonucleotide was used either as a probe or as a competitor in
electrophoresis mobility shift assay (EMSA) experiments. Nuclear
extracts were isolated from human dermal fibroblasts using a small
scale preparation (17), aliquoted in small fractions to avoid
repetitive freeze-thawing, and stored at
80 °C until use. The
protein concentration in the extracts was determined using a commercial
assay kit (Bio-Rad). Nuclear extracts (5 µg) were incubated for 20 min on ice in binding reaction buffer (10 mM HEPES-KOH, pH
7.9, at 4 °C, 4% glycerol, 40 mM KCl, 0.4 mM EDTA, and 0.4 mM dithiothreitol) in the
presence of 1 µg of poly(dI-dC), prior to the addition of
[32P]5'-end-labeled oligomers (0.05-0.1 pmol, 2-6 × 104 cpm) for another 20 min of incubation at 4 °C.
For competition experiments, a 1-60-fold molar excess of unlabeled DNA
was added to the binding reaction. For supershift experiments, nuclear
extracts were incubated overnight with antisera prior to the binding
reaction. Sp1 and c-Jun/AP-1 antibodies were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). The pan-Smad antibody 367 was
described previously (21). Samples were then separated by
electrophoresis on a 4% polyacrylamide gel in 0.5 × Tris
borate-EDTA buffer at 200 V for 2 h at 4 °C, fixed for 1 h
in 30% methanol, 10% acetic acid, vacuum-dried, and
autoradiographed.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Up-regulates Human COL7A1 Expression at a Transcriptional
Level--
We and others have previously demonstrated that TGF-
is
a potent inducer of type VII collagen gene expression in dermal
fibroblasts, as determined at both protein and mRNA levels (10,
11). To investigate whether TGF-
up-regulates COL7A1 gene expression at the transcriptional level by activation of the promoter, human dermal fibroblast cultures were transiently transfected with several 5'-end deletion/CAT reporter gene constructs spanning the COL7A1 promoter region from positions
722 to +92, relative to the
transcription start site, +1 (8). Cells were subsequently treated with
TGF-
(10 ng/ml) for 40 h, at which point CAT activity
(representing COL7A1 promoter activity) was determined. As shown in
Fig. 1A, a stimulatory effect
of TGF-
was observed with constructs
722COL7A1/CAT (~4.4-fold
induction) and
524COL7A1/CAT (~5.3-fold induction), indicating that
the up-regulation of type VII collagen gene expression by TGF-
occurs, at least in part, at the transcriptional level through
activation of the promoter. Subsequent 5'-deletion to position
456
abolished the COL7A1 promoter responsiveness to TGF-
, which was
similarly lost when further 5'-deletions extended to positions
396
and
230 (Fig. 1A). These data indicate that the DNA
sequences between residues
524 and
456 of the COL7A1 promoter are
essential in providing TGF-
responsiveness in fibroblasts.
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Fig. 1.
Effect of TGF- on the COL7A1
promoter activity. A, confluent fibroblast cultures were
transiently transfected with various 5'-deletion constructs of the
human COL7A1 promoter linked to the CAT gene and treated with TGF-
,
as described under "Materials and Methods." After measuring
-galactosidase activity in each extract, identical amounts were used
for constructs
722 and
524, whereas extracts corresponding to five
times the
-galactosidase activity were used for constructs
456,
390, and
230 to obtain a clearly detectable activity, as the basal
activity of these shorter constructs is significantly lower (see Ref.
8). CAT activity was assayed using [14C]chloramphenicol
as a substrate, as described under "Materials and Methods."
B, fibroblast cultures were transfected with either
524COL7A1/CAT or
524mCOL7A1/CAT promoter constructs. The GT box
mutation in the
524m construct prevents Sp1 binding and reduces
promoter activity by ~80% (8). CAT assays were normalized for
identical
-galactosidase activity in each sample. In each
panel, a representative autoradiogram is shown. Quantitative
results are the mean ± S.D. of six (A) or three
(B) separate experiments, each performed with
duplicate samples. Values are expressed as relative CAT
activity. AC and C indicate the acetylated and
unacetylated forms, respectively, of
[14C]chloramphenicol.
TGF- Induces the Rapid Formation of a Nuclear Protein-DNA
Complex Binding to the
524/
444 Fragment of COL7A1 Promoter--
To
determine whether the region of COL7A1 promoter shown above to confer
TGF-
responsiveness to a CAT reporter construct contained a putative
TGF-
specific cis-element, EMSAs were performed using a
radiolabeled 81-bp oligonucleotide spanning the region located between
residues
524 and
444, encompassing the entire TGF-
-responsive
COL7A1 promoter region. In the first set of experiments, fibroblast
cultures were incubated without or with TGF-
(10 ng/ml) for 24 h prior to nuclear extract preparation. No difference in the EMSA
pattern could be detected between nuclear extracts from control
cultures and those from TGF-
-treated cultures (not shown). In the
second set of experiments, fibroblast cultures were treated with
TGF-
(10 ng/ml) and nuclear extracts were prepared at several times
thereafter, between 1 min and 3 h. As shown in Fig.
2A, incubation of the nuclear
extracts with the
524/
444 oligonucleotide resulted in the formation
of two major, TGF-
-independent DNA-protein complexes, identified as
shifts 1 and 2, and present in nuclear extracts from either control or
TGF-
-stimulated fibroblasts. These two bands have been previously
shown to contain the transcription factor Sp1 (8). An additional
complex migrating between complexes 1 and 2 was induced as early as 15 min after TGF-
stimulation, persisted with a similar intensity until
the 1-h time point, and disappeared at 3 h. This new complex was
not present in nuclear extracts from control fibroblast cultures and
will be referred to as the TGF-
-specific band (TBSB).
|
Formation of the TBSB Requires Two Distinct Sites on the
524/
444 Region of COL7A1 Promoter--
The next set of experiments
was designed to further refine the cis-acting element(s)
responsible for the appearance of the TBSB. Toward this end, a series
of oligonucleotides was generated, representing a stepwise deletion
from either the 5'- or 3'-ends of the
504/
444 promoter fragment.
Their sequences and relative positions are depicted in Fig.
3A. First, EMSA experiments
were performed with nuclear extracts from fibroblast cultures treated for 30 min with TGF-
, using each of the various stepwise deletion oligonucleotides as probes. As expected from the competition
experiments presented above, the
504/
444 fragment efficiently bound
the TBSB (Fig. 3B, lane 2). 5'-End
deletion from nucleotide
504 to nucleotide
496 did not affect TBSB
formation with oligonucleotides extending to position
444 in 3'
(lanes 4 versus lane
2). However, further 5'-end deletion to residue
490 or
3'-end deletion to residue
453 resulted in complete loss of
TGF-
-induced binding activity (lanes 6 and
8, respectively). Three conclusions could be drawn from
these experiments. First, the minimal COL7A1 promoter fragment capable
of binding the TBSB is
496/
444. Second, it is likely that two
distinct sites, one located within the sequences surrounding
nucleotides
496/
490 at the 5'-end of the TBRS and the other between
nucleotides
453/
444 at the 3'-end of the TBRS, are simultaneously
required for providing the TGF-
-induced binding activity. Third,
deletion of the Sp1 binding GT box between
512 and
505 did not
influence the formation of the TBSB, further indicating that the GT box
is not involved in TGF-
response. Finally, despite the removal of
the GT box, residual Sp1 binding activity was still observed,
suggesting the presence of a secondary Sp1 binding sequence within the
490/
453 DNA fragment of COL7A1 promoter. The latter observation was
confirmed in two subsequent experiments. First, recombinant Sp1 protein
was capable of binding this fragment, as determined in gel mobility
shift assays, and second, an antibody specifically directed against Sp1
supershifted the weak protein-DNA complexes 1 and 2 generated with the
504/
444 COL7A1 probe (not shown).
|
Identification of Smad as Part of the TGF--induced DNA-Protein
Complex--
We next attempted to delineate the signaling pathway by
which TGF-
induces the formation of the TBSB. For this purpose,
fibroblast cultures were first incubated with several inhibitors of
different signaling pathways, prior to TGF-
stimulation (30 min) and
subsequent nuclear protein preparation. All inhibitors were tested at
three concentrations, which have been shown in previous publications to
effectively block their target pathways. Protein kinase C inhibitors (GF10923X, staurosporine, calphostin, H7), phospholipase C inhibitors (D609, U73122), tyrosine kinase inhibitors (genistein, tyrphostin 51),
the tyrosine phosphatase inhibitor sodium orthovanadate, mitogen-activated protein kinase pathway inhibitors (SB203580, PD
98059), calcium ionophore, pertussis and cholera toxins, and okadaic
acid all failed to block TBSB formation (not shown). This lack of
effect from all inhibitors tested suggests a rapid mechanism that may
be triggered directly through the TGF-
receptors, without a complex
cytoplasmic cascade of events, involving the several pathways listed
above. In this context, Smad proteins were recently identified as
immediate-early response factors, which are translocated into the
nucleus following their phosphorylation by TGF-
receptor type I
(reviewed in Refs. 22-25).
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DISCUSSION |
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Our studies of the human COL7A1 promoter reported here demonstrate
that this gene is an immediate-early target of TGF- transcriptional activation in dermal fibroblasts, with evidence of formation of a
TGF-
-specific transcriptional complex within 11 min after TGF-
addition in a ligand-dependent manner. Smad proteins have
recently been identified as critical intracellular mediators of TGF-
family-induced signals (reviewed in Refs. 22-25). Upon ligand binding
to TGF-
type I and II receptors, two Smad isoforms, Smad2 and Smad3,
are recruited to the type I receptor where they undergo phosphorylation on conserved serine residues in the C terminus. Following activation, these proteins associate with the tumor suppressor Smad4/DPC4 and are
translocated to the nucleus where they presumably act in
transcriptional complexes (reviewed in Refs. 22-25). In our experiments, supershift assays using a pan-Smad antibody (21) show that
the protein-DNA complex, TBSB, contains one or more Smad proteins.
Based on signaling specificity, this complex likely contains Smad2 or
Smad3 along with Smad4.
The method of transcriptional activation by Smads has recently begun to
be elucidated. Chen et al. (26) identified a DNA-binding protein of the forkhead family (FAST-1) that specifically and inducibly
interacts with an activin-responsive element (ARE) in the promoter of
the Xenopus homeobox gene Mix.2 (27). FAST-1 also
interacts with Smad2 and Smad4/DPC4 in the ARE binding complex (28).
Reconstitution of this system in mammalian cells demonstrates that
efficient DNA binding and transcriptional activation require FAST-1,
Smad2, and Smad4 in the same complex (29). Our data represent the first
example of a mammalian promoter directly regulated by interactions with
endogenous Smad proteins and as such make COL7A1 the first example of
an immediate-early gene regulated by TGF- in a
Smad-dependent fashion.
Although the cis-elements necessary for interaction
with Smad transcriptional complexes are not known, some architectural constraints may be deduced from comparison of the TBRS in the COL7A1
promoter with the ARE from the Mix.2 gene. Chen et
al. (26) identified a direct 6-bp AAATGT repeat separated by 11 bp, and this sequence was used to clone the DNA binding protein FAST-1.
Mutation of either element of this repeat disrupts DNA binding by
FAST-1 (26). Similarly, the COL7A1 element contains two 5-bp ATGGC
repeats, two adjacent CAGA repeats in the 5'end and two pairs of 4-bp
repeats, CCCA and ACAG. Deletion studies suggest that two elements are
necessary for Smad/DNA interaction, as both the COL7A1 490/
444 and
496/
453 probes failed to bind the TGF-
-inducible complex,
whereas the
496/
444 fragment was the minimum required to generate a
gel-shifted band. This suggests a mechanism similar to that observed
with the Mix.2 ARE, with two separated elements required for
full activity (26). The nature of the exact cis-elements
required for DNA-Smad interaction is currently under investigation.
Whether a Smad protein binds the COL7A1 promoter directly cannot be
ascertained from our data. The Drosophila Smad1 homologue,
the mad gene product, can bind elements in the
vestigial promoter directly, and a consensus Mad binding
sequence (GCCGnCGc) that can bind recombinant Mad protein has been
identified (30). Similarly, the tumor suppressor Smad4 can bind DNA
directly (31), but this has not been convincingly demonstrated in an
endogenous complex. In the COL7A1 promoter, an accessory DNA binding
protein may be required, as in the Mix.2 ARE, or a Smad or
Smads may bind the TGF-
-responsive elements without additional
binding proteins. Experiments are under way to explore these
possibilities.
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ACKNOWLEDGEMENT |
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The expert technical assistance of Ying-Jee Song is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants 29-AR43751 (to A. M.) and RO1-AR41439 and T32-AR07651 (to J. U.) and by a Research Career Development Award from the Dermatology Foundation (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, 233 South 10th St., Rm. 430, Philadelphia, PA
19107. Tel.: 215-503-5775; Fax: 215-923-9354; E-mail:
mauviel1{at}jeflin.tju.edu.
1
The abbreviations used are: TGF-,
transforming growth factor-
; ARE, activin-responsive element; CAT,
chloramphenicol acetyltransferase; EMSA, electrophoresis mobility shift
assay; TBSB, TGF-
-specific band; TBRS, TGF-
-responsive sequence;
bp, base pair(s).
2 R. J. Lechleider and A. B. Roberts, unpublished data.
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REFERENCES |
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