(Received for publication, July 26, 1996, and in revised form, October 18, 1996)
From the Perlecan, a modular heparan sulfate
proteoglycan of basement membranes and cell surfaces, plays a crucial
role in regulating the assembly of extracellular matrices and the
binding of nutrients and growth factors to target cells. To achieve a
molecular understanding of perlecan gene regulation, we isolated the
5 Perlecan (1), a modular proteoglycan carrying primarily heparan
sulfate side chains, is present in virtually all vascularized tissues
(2, 3). This proteoglycan is expressed either as an integral component
of basement membranes or diffusely in connective tissues as diverse as
ovarian stroma, skin, and cartilage (4, 5). Perlecan epitopes are
distributed along the sinusoidal spaces of the liver, spleen, bone
marrow, and lymphoreticular system including thymus, tonsils, and lymph
nodes (4). The latter suggests that perlecan may be important for the
normal development and function of the bone marrow and lymphoreticular system (6). Analysis of the primary structure of human perlecan reveals
an assembly of five consecutive domains with homology to molecules
involved in the control of cell proliferation, lipoprotein uptake, and
adhesion (1, 7). This molecular architecture suggests that perlecan may
play a variety of biological functions in different tissue locations
(8). For example, perlecan can bind several growth factors via its
heparan sulfate side chains (9, 10), while the protein core perlecan
can bind nidogen (11) and various integrins (12, 13). Recently, we
provided direct evidence that basic fibroblast growth factor binds to
heparan sulfate chains attached to domain I and that the concerted
action of heparanases and proteases could modulate the bioavailability of this growth factor in vivo (14). The perlecan-mediated
induction of angiogenesis is supported by the observation that
metastatic melanoma cells exhibit a marked induction of this gene, and
this increase correlates with an abundant deposition of perlecan
proteoglycan in the neovascularized tumor stroma (15). In addition,
perlecan binds to fibronectin (16), Perlecan expression is up-regulated by nanomolar concentrations of
TGF- We previously reported the complete genomic organization of the human
perlecan HSPG2 gene, including 0.77 kb of the 5 Radionucleotides
[ Deletion constructs cloned upstream of
the CAT reporter gene within the pUC or pBluescript (pBS) vector
(Stratagene) were generated as described before (28). The 2.5-kb
5 Transient
transfections of cell types were performed by the calcium phosphate
procedure essentially as described before (28, 29). Briefly,
subconfluent cells in 75-cm2 flasks were trypsinized,
washed, and co-transfected in suspension with 20-40 µg of perlecan
promoter-CAT constructs and 10 µg of pSV- Total RNA was isolated using standard procedures
(31) and analyzed by Northern blotting as described before (29). Human perlecan cDNA, clone HS1, encompassing domain III (32) was used as
a probe, or the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used for normalization of the values. Both probes were radiolabeled (~109 cpm/µg of DNA) by random
priming (33) using [ Nuclear extracts from fibroblasts treated with or
without TGF- Double stranded
oligonucleotides were radiolabeled with 32P using the T4
polynucleotide kinase, purified by acrylamide gel electrophoresis, and
eluted overnight. A series of oligonucleotides (listed in Table I) were
tested in competition assays including the TGF-
Structural similarity of TGF- Department of Pathology,
Department
of Dermatology and Cutaneous Biology, and the ** Jefferson Institute of
Molecular Medicine,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-flanking region and investigated its functional promoter activity
and its response to cytokines. Transient cell transfection assays,
using plasmid constructs harboring the perlecan promoter linked to the
chloramphenicol acetyltransferase reporter gene, demonstrated that the
largest ~2.5-kilobase construct contained maximal promoter activity.
This promoter region was functionally active in a variety of cells of
diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5
deletion analyses demonstrated that the
461-base pair (bp) proximal promoter retained ~90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-
induced 2-3-fold perlecan mRNA and protein core levels in normal human skin
fibroblasts, and this induction was transcriptionally regulated; in
contrast, tumor necrosis factor-
had no effect and was incapable of
counteracting the effects of TGF-
. Using additional 5
deletions and
DNase footprinting analyses, we mapped the TGF-
responsive region to a sequence of 177 bp contained between
461 and
285. This region harbored a 14-bp element similar to a TGF-
-responsive element present in the promoters of collagen
1(I),
2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-
-responsive element bound specifically to TGF-
-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.
-interferon (17), and
TGF-
1 (18), and recent evidence indicates
that perlecan can be adhesive for fibroblasts, endothelial cells (19),
and chondrocytes (5) while being anti-adhesive for hematopoietic cells
(19). These opposing functions are not surprising given the
multipurpose structure of the protein core and the complexity of its
potential post-translational modifications. The latter may result in
heparan sulfate side chains either alone (20) or in combination with
chondroitin sulfate (21), fatty acylation (22), or the total lack of
glycanation of the protein core which can be produced as a
glycoprotein (5, 23).
in human colon carcinoma cells (24) and murine uterine
epithelial cells (25). TGF-
also stimulates the synthesis of basic
fibroblast growth factor-binding heparan sulfate proteoglycans in mouse
3T3 cells (26). Moreover, it is quite evident that perlecan is a key
regulator of the complex interactions that take place during
neovascularization, and that the interplay amongst TGF-
, basic
fibroblast growth factor, and perlecan is biologically relevant (3,
14).
-flanking region (27). In the current investigation we cloned and sequenced 2.5 kb of the upstream region, and, by using transient cell transfection assays and stepwise 5
or internal deletions, we demonstrate functional promoter activity of the perlecan gene. The perlecan promoter was
transcriptionally activated by TGF-
but was unresponsive to TNF-
.
A TGF-
-responsive element was mapped to a 14 bp sequence located in
the proximal promoter region by DNase footprinting and electrophoretic
mobility shift assays. The perlecan TGF-
-responsive element bound
specifically to TGF-
-inducible nuclear proteins which contained NF-1
members of transcription factors. Collectively our results indicate
that perlecan is up-regulated by TGF-
via a transcriptionally
mediated control of perlecan promoter and that these effects could be
important in the modulation of this proteoglycan during angiogenesis,
tissue remodeling, and tumor formation.
Materials and Cell Cultures
-32P]dCTP, [
-32P]ATP and
[
-32P]ATP (3000-5000 Ci/mmol; 1 Ci = 37 Gbq) and
[14C]chloramphenicol (~100 mCi/mmol) were obtained from
Amersham Corp. Restriction endonucleases were purchased from Promega
(Madison, WI). Human recombinant TNF-
was purchased from Boehringer
Mannheim. Human recombinant TGF-
2 was a generous gift from Dr. David
Olsen (Celtrix Laboratories, CA). HeLa, WiDr colon carcinoma, Saos-2 osteosarcoma, HL-60 promyelocytic leukemia, HT1080 fibrosarcoma, and
NIH-3T3 fibroblastic cells were obtained from the American Type Culture
Collection. Mouse M2 melanoma cells were obtained from Dr. I. J. Fidler
(Houston, TX). Human diploid fibroblast cultures, established from
neonatal foreskins, were utilized at passages 3-8. The cell cultures
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum (Life Technologies, Inc.), 2 mM
glutamine, and 100 units/ml penicillin.
-flanking region linked to exon 1 (86 bp) was subcloned into the pBS
vector and then subcloned into the respective sites in the pUC-CAT
vector. Shorter constructs were generated by deletion of the 2.5 kb
construct using various restriction endonucleases. Constructs
1800
and
2500/
1800 were generated by digestion of the
2500 construct with HindIII and EcoRV, which deleted ~1800 and
~700-bp fragments between
2500 and
1800. The asymmetric
distribution of SmaI sites in the
2500 construct allowed
the utilization of partial digestion of the construct thereby
generating various 5
and internal deletion products. These constructs
were subsequently self-ligated and sequenced to confirm the correct
orientation.
-galactosidase plasmid to
provide an internal standard for normalization of the values. The cells
were incubated for 48 h, washed with Hanks' balanced salt
solution (Ca2+- and Mg2+-free), and incubated
in Dulbecco's modified Eagle's medium for an additional 12 h.
The cells were then washed again and assayed for
-galactosidase
activity (29). CAT assays were performed as described before (30). The
products were resolved on preslotted thin layer chromatography plates
in a chloroform/methanol (95:5) mobile phase and subjected to
autoradiography. To quantify the acetylated
[14C]chloramphenicol, the autoradiograms were subjected
to scanning laser densitometry and computer integration. To study the
transcriptional regulation by TGF-
and TNF-
, minor variations to
the protocols described above were made. Essentially, cells were
treated as above except that the cells were placed in Dulbecco's
modified Eagle's medium containing 1% fetal calf serum 4-5 h before
the addition of growth factors. In experiments without growth factors, the cells were placed in DMEM containing 1% fetal calf serum. After
40 h of additional incubation, the cells were rinsed with phosphate-buffered saline and harvested. Aliquots corresponding to
identical
-galactosidase activity were used for each CAT assay. After autoradiography, the plates were cut and counted (29).
-32P]dCTP. The
[32P]cDNA-mRNA hybrids were visualized by
autoradiography, and the steady-state levels of perlecan or
glyceraldehyde-3-phosphate dehydrogenase were quantitated by laser scan
densitometry and computer integration, using various exposures to
guarantee a linear range. For immunoblotting, human skin fibroblasts
were grown to confluence, washed and incubated for 48 h in
serum-free medium in the presence or absence of either TGF-
or
TNF-
. At the end of each incubation, aliquots of media were
processed for slot immunoblotting (6). Briefly, scalar dilutions of
media were blotted into nitrocellulose strips using a Minifold II
(Schleicher and Schüll); the strips were blocked with 0.05%
Tween 20, 4% nonfat dry milk and incubated with the monoclonal
antibody 7B5 directed against domain III of human perlecan (4).
Following several washes, the membranes were incubated with horseradish peroxidase-labeled goat anti-mouse IgG, washed again, and incubated with the chemiluminescence substrate reagent (15). The membranes were
exposed to radiographic films for 0.5-1 min and quantified by laser
scanning densitometry.
(10 ng/ml) for 18-24 h or from HeLa cells were
prepared essentially as described before (34). Protein concentration
was determined by a colorimetric method (35). At the end of each
purification, aliquots were snap-frozen in liquid nitrogen and stored
at
80 °C until analyzed. DNase footprinting analysis was performed
to map the nuclear protein binding sites in the proximal promoter
region. To this end, the
461 promoter CAT construct was labeled by
Klenow with [32P]dCTP on the coding strand at the unique
RsrII site, located at
118. Briefly, the
RsrII-linearized plasmid was isolated by agarose gel
electrophoresis, labeled to high specific activity with Klenow enzyme
(New England Biolabs Inc.), purified by column chromatography, and
heated at 65 °C to inactivate the enzyme. The fragments were
purified and subjected to HindIII digestion, and the
released 344-bp 32P-labeled fragment was separated from the
~5-kb vector by agarose gel electrophoresis. The
HindIII-RsrII fragment was identified by exposing
the gels to an x-ray film, electrophoresed for 10 min onto DEAE paper
slotted in the gel, and eluted in TAE buffer (40 mM Tris
acetate, 1 mM EDTA, pH 7.2) overnight. The labeled DNA
fragments (2-4 × 104 cpm) were first incubated for
15 min on ice with 5-10 µg of nuclear extracts in a buffer
containing 1 µg of poly(dI-dC), 10 mM Tris, pH 8.0, 4 mM MgCl2, 1 mM CaCl2, 2 mM dithiothreitol, 1 µg of bovine serum albumin, and 0.1 M KCl. The solutions were then subjected to DNase I
digestion (100 milliunits), terminated by 3 volumes of a solution
containing EDTA (10 mM) and yeast tRNA, precipitated with
ethanol, resuspended in a denaturing buffer, and analyzed on a
denaturing 6% gel (36). Radioactive DNA molecular mass markers were
run in parallel (29).
responsive element
(T
RE), a 14-bp sequence located between
301 and
314, four
mutated T
REs, a 14-bp TGF-
-responsive sequence of the rat
1(I)
collagen promoter, and consensus oligodeoxynucleotides for the NF-1 and
AP-2 transcription factors. The labeled probes were incubated for 15 min in a buffer containing 100 mM Hepes, pH 7.9, 1 mM EDTA, 0.2 M NaCl, 40 mM
MgCl2, 20 mM dithiothreitol, 20 mM
spermidine, 1-2 µg of poly(dI-dC), and 4% glycerol (29), with or
without molar excess of oligonucleotide competitors. Nuclear extracts
(10-20 µg) from control or TGF-
-treated fibroblasts or HeLa cells
were added to the mixtures and incubated for an additional 15 min. The
protein-DNA complexes were separated on 6% polyacrylamide gels (20:1;
acrylamide to bisacrylamide) in 22 mM Tris borate, 22 mM boric acid, and 0.5 mM EDTA (37). The gels
were fixed, dried under vacuum, and exposed to x-ray films. Protein-DNA
complexes were removed from the dried gels, and the radioactivity was
measured in a scintillation counter.
-responsive elements in various
mammalian promoters
responsive element (T
RE) in the
human perlecan promoter vis á vis T
REs previously shown to be
capable of mediating TGF-
responsiveness in transient cell transfection or DNA binding assays, with the exception of the human
growth hormone where no TGF-
induction was investigated (54) and for
the human
2 integrin for which no functional analysis was
provided (44). The 5
and 3
homologous sequences are
boldface. The binding sites for NF-1 and AP-2 transcription
factors are boldface and underlined. Gaps are
introduced to facilitate alignment. The T
RE consensus is based also
on the mutational analysis of the sequence as investigated by
electrophoretic mobility shift assays (cf. Figs. 8, 9, 10).
Gene
Position
Sequence
Reference
Human
perlecan
314
5
-TGGCCCGGCGGCCC..-3
Present
study
Rat
1 (I)
collagen
1643
5
-TGCCCACG..GCCAAG-3
37
Mouse
2 (I)
collagen
308
5
-TCGCCCTT..GCCAAG-3
52
Human
elastin
138
5
-TCCCCCAG..GCCTCC-3
53
Human type I plasminogen activator
inhibitor
560
5
-TGGCTGCAT.GCCCT -3
55
Human growth
hormone
285
5
-TGGCCTGCG.GCCAG.-3
54
Human
2
integrin
235
5
-TGGCTAGGGCGCCA..-3
44
NF-1
consensus
5
-
TTGAA.
AA- 3
58
AP-2
consensus
5
-C
CGC..
CCGT-3
38
T
RE
consensus
5
-TGGCC.N3-5.GCC. -3
By screening a chromosome 1-specific phage library with a
human cDNA probe encompassing the 5 region of the perlecan gene, we isolated about 4.5 kb of the sequence 5
to exon 1. A
computer-assisted analysis, using the Signal and Eukprom programs from
the GCG and PCGENE packages, revealed that the 5
-flanking region (Fig.
1) lacked canonical TATA or CAAT boxes but contained
several features of a promoter including: (a) a relatively
high GC content, primarily evident in the proximal region, with 80%
GpC, typical of CpG islands, and (b) the presence of
numerous cognate cis-acting elements and palindromic
inverted repeats. These features are typical of housekeeping and growth
factor-encoding genes that characteristically lack TATA or CAAT boxes
and contain multiple transcription initiation sites, as in the human
HSPG2 gene (27). The majority of the cognate
cis-acting elements were clustered in the first 1.5 kb as
related to the major transcription initiation site (Fig. 1). The
proximal region contained four GC boxes and 15 consensus hexanucleotide binding sites for the zinc finger transcription factor Sp1, 5 of which
were located in the first exon. Another striking feature of the
perlecan promoter was the presence of numerous AP-2 motifs (38), eight
residing in the first 1.5 kb and two in the most distal area. In
addition, numerous AP-2 binding sites were scattered throughout the
noncoding strand (not shown). These potential binding sites for the
AP-2 transcription factor possess the following two important features:
(a) their binding and transcriptional activity are inhibited
by SV40 T antigen, and (b) they confer phorbol ester
12-O-tetradecanoylphorbol-13-acetate and cAMP inducibility (39). Of note, SV40 large T antigen inhibits transcription of perlecan
in renal tubular epithelial cells (40), and perlecan expression is
markedly up-regulated by
12-O-tetradecanoylphorbol-13-acetate in colon cancer (41) or
K562 leukemia (6) cells. The proximal promoter region also contained
three palindromic inverted repeats in close proximity of each other
(between
438 and
204) that, by forming secondary structure, could
influence the regulation of perlecan gene expression. The perlecan
promoter contained several motifs that bind transcription factors
involved in hematopoiesis (42) including two PEA3 motifs, primary
targets for signal transduction that bind to Ets members of
oncogene, one Ets-1 and one PU.1 box that confers
transcriptional activation to B lymphocytes (43), and nine GATA-1
motifs that are involved in erythrocyte differentiation (42). In
addition, multiple GATA-1 sequences were present in the noncoding
strand (not shown). The adjacent binding sites for GATA and
Ets transcription factors at
1830 and
1802,
respectively, are similar to those described in the human
2 integrin promoter (44) and suggest the possibility of
either cooperative interaction or competitive inhibition. The distal
promoter region contained a binding site for NF-kB, a factor
that has been involved in interleukin-1 stimulation of transcription in
a variety of genes (39). Two fully conserved CTF-NF-1 elements at
position
2524 and
1947 in a gene that lacks both TATA and CAAT box
sequences are very close to those identified in the human
2 integrin (44) and in a variety of genes including the
chicken
globin, mouse SPARC and a silencer site in the mouse
2(I) collagen gene (45). In these genes, the CTF-NF1 sequences serve
as negative regulatory elements which suppress gene transcription in a
cell type-specific fashion (45).
The 5
To prove
that the sequence upstream to exon 1 can exhibit transcriptional
activity, we transfected a full-length promoter-CAT construct harboring
a ~2.5-kb sequence and 86 bp of exon 1, into various human cell lines
of epithelial or mesenchymal origin and also into two murine cell lines
(Fig. 2). When the values were normalized on
-galactosidase activity, an internal control for changes in
transfection efficiency, functional promoter activity was detected in
all the cells tested with maximal activity in the WiDr colon carcinoma
cells (Fig. 2B). This is consistent with the fact that these tumor
cells express high levels of this proteoglycan (20, 23). The activity
of the perlecan promoter in such diverse cell types is in harmony with
the widespread tissue distribution of perlecan (4). The activity of
perlecan promoter in HL-60 promyelocytic leukemia cells (Fig. 2B)
corroborates our previous study which has shown that perlecan is
expressed in lymphoreticular cells (6) and further emphasizes the
importance of the numerous motifs that could bind cognate transcription
factors involved in hematopoiesis (see above). Relatively high perlecan
promoter activity was also observed in murine cells, e.g.
NIH-3T3 fibroblasts and M2 melanoma cells (Fig. 2B,
filled bars), indicating that the human perlecan promoter is
active in another species. Taken together, these findings validate the
widespread distribution of perlecan demonstrated before by
immunohistochemical (4, 7, 46) and in situ hybridization (4,
7) analyses and further suggest that the levels of perlecan gene
expression may be regulated by tissue-specific factors.
Deletion Analysis of the Perlecan Gene Promoter
To
investigate in more detail the functional properties of the perlecan
gene promoter, we performed reporter gene analysis using various
perlecan promoter-CAT fusion plasmids harboring either 5 stepwise or
internal deletions (Fig. 3A). In these experiments we
used HeLa cells because of their ease of transfection and because we
found that they express perlecan,2 a fact
that was confirmed by the activity of the full-length promoter-CAT
construct in transient cell transfection assays (Fig. 2). After
normalization on
-galactosidase activity, several perlecan promoter
constructs exhibited considerable CAT activity. The relative CAT
activity of nine constructs based on data from several
(n = 6-14) independent experiments is summarized in
Fig. 3B. The largest construct, harboring the 2.5 kb of 5
-flanking
region, contained maximal promoter activity set arbitrarily to 100%.
Shortening the upstream sequence from
2500 to
1800 bp did not
produce significant changes in CAT activity. However, three internal
deletion constructs (
2500/
283,
2500/
560, and
2500/
1800)
showed a marked reduction of CAT activity. Indeed, when these values
were corrected for nonspecific base line activity expressed by the two
promoterless control plasmids (pBSCAT and pUCCAT), the internal
deletions showed essentially complete loss of activity. Consistent with
these data, the
461-bp construct maintained 80-90% promoter
activity as compared with that detected in the two longest constructs,
respectively. Additional reduction was observed with other constructs
(Fig. 3B). Sequence analysis of this proximal region
revealed, in addition to the multiple AP-2 sites and three palindromic
inverted repeats, a regulatory element located at position
450 that
consisted of a 5
-GTGAGCGTG-3
sequence with homology to the consensus
sequence of the DE1 element 5
-TGACGGTG-3
of the mouse
A-crystallin
gene (47). This cis-acting motif resembles the
cAMP-responsive element sequence (ATF/CREB), 5
-GTGACGT(A/C)(A/C)-3
,
and behaves as a functional CREB site (47). Therefore, it is possible
that this region in the perlecan promoter may be connected with a cAMP
transduction. Present in the most proximal promoter region (
132-bp
construct) were four GC boxes and several binding sites for Sp1, a well
characterized transcription factor that binds GC boxes and stimulates
transcription in promoters that contain these sites (39), including the
murine syndecan 1 (48) and thrombospondin 1 (49) genes. These results indicate that the 5
-flanking region of the perlecan gene can act as a
functional promoter and contains a complex array of
cis-acting elements necessary for driving the expression of
the perlecan gene in both epithelial and mesenchymal cells.
Induction of Perlecan Gene Expression in Human Skin Fibroblasts by TGF-
In the next series of experiments, we
wished to investigate the modulation of perlecan gene expression by
TGF- and TNF-
. We utilized normal human diploid skin fibroblasts
because, in contrast to HeLa cells, these cells respond to various
cytokines and their proteoglycan makeup has been well investigated (7, 29, 50). To this end, post-confluent skin fibroblasts were preincubated
for 5 h with 1% fetal calf serum and then incubated for 24 h
in the absence or presence of TGF-
(5 ng/ml) or TNF-
(5 ng/ml),
either alone or in combination. Total RNA was then extracted and
analyzed for perlecan or glyceraldehyde-3-phosphate dehydrogenase gene
expression (Fig. 4). While TGF-
up-regulated the
steady state levels of perlecan mRNA (Fig. 4, A and
B, lane 2), TNF-
had no significant effects
(Fig. 4, A and B, lane 3). Interestingly, TNF-
did not block the up-regulation of perlecan gene
expression induced by TGF-
(Fig. 4, A and B,
lane 4).
To verify that the increased perlecan mRNA levels correlated with
an increased proteoglycan synthesis, we performed immunoblotting experiments using a monoclonal antibody directed against domain III of
perlecan (4). A ~2-fold increase in perlecan protein core
biosynthesis in the presence of TGF-, but no effect in the presence
of TNF-
, was observed (Fig. 5). Because most of the synthesized perlecan (>90%) is released as soluble proteoglycan (3),
these results indicate that the transcriptional effects of TGF-
are
also seen at the protein level, albeit at a degree lower than that
observed at the mRNA level. Our results are in agreement with the
stimulation of perlecan biosynthesis and secretion by TGF-
observed
in other cellular systems including colon carcinoma (24), uterine
epithelial (25), and 3T3 fibroblastic (26) cells.
Identification of a TGF-
The results presented above suggested that the promoter
of the perlecan gene harbored sequences that could respond to TGF-. To characterize these putative TGF-
-responsive element(s), we initially tested the largest construct of 2.5 kb for sensitivity to the
cytokine. In this series of experiments, the cells were transfected
with the promoter CAT-containing plasmid and subjected to subsequent
culture in the presence or absence of the cytokine. The results (Fig.
6) showed that the entire promoter of human perlecan was
transcriptionally activated by about 2.6-fold upon treatment with
TGF-
. To delineate more accurately the TGF-
-responsive region, we
made additional promoter-CAT constructs and subjected them to the same
transient cell transfection assays in which the cells were cultured in
the presence or absence of the cytokine. The results showed that the
461-bp construct was markedly stimulated by TGF-
at a level even
higher than the largest (
2500-bp) construct, whereas all additional
5
deletions, including
285-,
189-,
158-, and
132-bp
constructs, lacked TGF-
responsiveness (Fig. 6). Taken together,
these results indicate that the proximal 461 bp of the human perlecan
promoter contains nearly all the regulatory elements to provide full
functional promoter activity and further show that a region of about
177 bp, located between
461 and
285 bp, harbors the TGF-
responsive sequence(s).
DNase Footprinting and Electrophoresis Mobility Shift Assay of the Proximal Promoter Region
The clear geographic demarcation of
TGF- action on the perlecan promoter indicated that the major
TGF-
activating element was located between
461 and
285 bp.
DNase I footprinting was used to discover cis-acting
elements in this relatively short sequence. The results showed at least
five distinct footprints, designated a-e (Fig.
7). Interestingly, similar results were obtained with
nuclear proteins isolated from either diploid fibroblasts (Fig.
7A, lane 3) or HeLa cells (Fig. 7A,
lane 4). However, only footprinting a was located
in the region which was responsive to TGF-
(Fig. 7B,
shaded sequence). Footprints b and e
harbored Sp1 binding sites, footprint d overlapped with an
AP-2 consensus sequence, whereas footprint c was adjacent to
an Sp1 binding motif.
A closer inspection of the 17-bp sequence footprinted by nuclear
proteins from either fibroblasts or HeLa cells, e.g.
footprint a in Fig. 7, showed that this sequence harbored a
14-bp motif that was very similar to a TGF--responsive element
(T
RE) previously observed in the promoter of rat
1(I) collagen
(37, 51), mouse
2(I) collagen (52), human elastin (53), human growth
hormone (54), human
2 integrin (44), and human
plasminogen activator inhibitor (55) genes (Table I).
All of these sequences, with the exception of the human
2 integrin, have been shown to be responsive to TGF-
by transient cell transfection, DNase footprinting, or electrophoretic
mobility shift assay analyses. Structurally, all of these T
REs are
characterized by internal NF-1 and AP-2 motifs, which may mediate the
action of TGF-
.
To examine the binding of the perlecan TRE to nuclear protein, a
double-stranded oligomer encompassing the perlecan T
RE was
end-labeled and incubated with equal amounts of nuclear proteins extracted from either untreated or TGF-
-stimulated (5 ng/ml for 24 h) fibroblast nuclei. Electrophoretic mobility shift assay analysis showed that the TGF-
-stimulated nuclear extracts contained >3 fold more protein binding activity than unstimulated samples and
that the binding was specific because it could be displaced by 10-fold
molar excess of unlabeled T
RE competitor (Fig. 8A). The results further showed that a comparable response was also obtained
with nuclear extracts from cells exposed to TGF-
for either 12 or
18 h (Fig. 8B).
An NF-1 Binding Site Mediates the TGF-
To investigate the specificity
of perlecan TRE, competition studies were performed with the
collagen
1(I) sequence, or consensus sequence encoding AP-2 and NF-1
binding sites (summarized in Table I). The results showed that
equimolar amounts of unlabeled collagen
1(I) T
RE (Fig.
9A, lane 5) or NF-1 (Fig.
9A, lane 7) were as effective as perlecan T
RE
(Fig. 9A, lane 4) in disrupting the protein-DNA
complex. In contrast, an oligonucleotide harboring the AP-2 consensus
sequence was ineffective (Fig. 9A, lane 6). To
investigate further the relationship between the perlecan and the
collagen
1(I) T
REs, we end-labeled the latter and performed competition studies. The results showed that, under identical electrophoretic conditions and nuclear extract concentrations, the
collagen
1(I) T
RE produced a more retarded complex (Fig. 9B, lane 2) and showed induction by TGF-
(Fig.
9B, lane 3). Interestingly, while unlabeled
collagen
1(I) T
RE completely disrupted the DNA-protein complex
(Fig. 9B, lane 5) equimolar concentrations of
perlecan T
RE reduced the complex by ~60% (Fig. 9B,
lane 4) and AP-2 was even less effective (Fig.
9B, lane 6). When the NF-1 consensus sequence was
end-labeled and tested with the same nuclear extracts, a DNA-protein
complex with a mobility identical to that detected with perlecan T
RE
was observed (Fig. 9C, lanes 1 and 2, respectively), further
suggesting an affinity of the perlecan T
RE for NF-1 member(s) of
transcription factors. In contrast, when recombinant AP-2 protein (Promega) was tested in the same electrophoretic mobility shift assay
system, no appreciable displacement of the specific DNA-protein complex
was observed (Fig. 9C, lane 5), further
corroborating the results obtained with competing AP-2 oligonucleotide.
Collectively, these studies support the conclusion that the perlecan
T
RE binds to transcription proteins of the NF-1 family, and that the
collagen
1(I) T
RE may contain additional transcription factors
that differ from those binding to the perlecan T
RE.
Finally, to investigate more directly the sequence specificity of the
perlecan TRE, we generated double-stranded oligomers harboring
various mutations in the 5
end, 3
end, or central region of the
perlecan T
RE (Fig. 10B). Three-bp substitutions in
either the 5
- or 3
-GCC region reduced almost completely the ability
of the perlecan T
RE to be an effective competitor of the specific
DNA-protein complex (Fig. 10A, lanes 5 and
6, respectively). In contrast, 3-bp or 4-bp substitutions in
the central domain of the core binding oligomer did not appreciably
influence the complex formation (Fig. 10A, lanes 3 and 4, respectively). These results are similar to
those obtained with the murine
2(I) collagen promoter where an
NF-1-like sequence is responsible for a 5-10-fold transcriptional
induction by TGF-
(52).
NF-1 was initially identified as a cellular factor that stimulates
in vitro replication of adenovirus DNA (56), but it was subsequently shown that this family of transcription factors plays a
role in both DNA replication and RNA transcription (57). Indeed, the
CAAT-binding/NF-1 family comprises at least six distinct nuclear proteins that are composed of heterologous subunits with binding affinity for imperfect palindromes of TGGC sequences separated by 6 or
7 bases, although they can also bind to a single TGGC sequence (57,
58). Flanking sequences, length and composition of the spacer regions
can greatly influence their binding affinity (57). Taking into account
the homology and the mutational analysis, thus, a consensus for TRE
(5
-TGGCC . N3-5...GCC-3
) could be generated
(Table I).
We have cloned and sequenced 2.5-kb of
5-flanking region of the human perlecan HSPG2 gene and
determined for the first time functional promoter activity using
transient cell transfection assays. The 5
-flanking region possesses
strong basal and inducible promoter activity and is thus assumed to
represent the promoter as it operates in vivo. The
ubiquitous distribution of perlecan correlates well with functional
promoter activity detected in cells with disparate histogenetic origin
and also of different species. This study has also identified a T
RE
in the proximal promoter region with affinity for members of the NF-1
family of transcription factors. Our data indicate that this sequence
is primarily responsible for the transcriptional activation of the perlecan gene by TGF-
. The results show a complex array of
regulatory factors for this proteoglycan and further indicate that the
level of constitutive expression in both epithelial and mesenchymal cells is likely guided by the proximal promoter region. The structural and functional characterization of the perlecan gene promoter will now
allow dissection of the complex regulation of this molecule during
normal development, tissue repair, and tumor growth.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L81166[GenBank].
We thank I. Eichstetter for excellent
technical assistance and D. Olsen for providing human recombinant
TGF-.