(Received for publication, May 13, 1996, and in revised form, February 18, 1997)
From the Departments of Medicine and Cell Biology, Division of Endocrinology and Metabolism, The University of Alabama at Birmingham, Birmingham, Alabama 35294
The epidermal growth factor receptor is vital for
normal development and plays a role in oncogenesis. The level of
activation of this receptor by transforming growth factor- (TGF-
)
is controlled, in part, by the rate of transcription of the TGF-
gene. In the characterization of the proximal TGF-
promoter by DNase
I footprinting, a 43-base pair element (
88 to
130 relative to the
transcription start site), designated T
RE I, was found that was
specifically protected by nuclear proteins from human mammary carcinoma
MDA468 cells. T
RE I was essential for the maximal expression of the TGF-
gene as indicated by deletion and mutagenesis analyses. T
RE
I consists of two cis-acting elements, a proximal
regulatory element (PRE,
89 to
103) and a distal regulatory element
(DRE,
121 to
128). Both elements were able to form specific
complexes with protein from MDA468 cell nuclear extracts and are
necessary for the full activity of the entire 1.1-kilobase pair TGF-
promoter. Competition and antibody studies determined that the DRE
contains a binding site for the transcription factor AP-2, while the
protein that binds to the PRE has yet to be identified. When linked
upstream to the heterologous herpes simplex thymidine kinase promoter, the T
RE I enhanced transcription up to 11-fold in MDA468 cells. Cotransfection of an AP-2 expression vector was able to activate transcription from the T
REI-TK construct in a
DRE-dependent manner. These results further our
understanding of how TGF-
transcription is regulated.
Transforming growth factor-
(TGF-
)1 is a polypeptide mitogen
belonging to the epidermal growth factor (EGF) family (1). TGF-
is
synthesized as a 160-amino acid transmembrane precursor from which the
50-amino acid mature peptide is released (2). Both the
membrane-anchored and soluble forms are biologically active through
interaction with cell surface EGF receptors.
First discovered in the media of retrovirally transformed fibroblasts
(3, 4), TGF- appears to play an important role in oncogenesis. It
has been found to be associated with many tumors such as mammary,
squamous, and renal carcinomas, melanomas, hepatomas, and glioblastomas
(5-7). When transfected with a TGF-
expression vector, certain cell
lines became transformed (8-10). Also, transgenic mice overexpressing
TGF-
often develop neoplastic lesions in the tissues to which
TGF-
overexpression has been directed (11-14). In many cases, tumor
cells coexpress TGF-
and the EGF receptor, suggesting an autocrine
mechanism of growth stimulation (15). That TGF-
is also a potent
inducer of angiogenesis in vivo (16) suggests that host
support for the tumor might also be induced through the expression of
this growth factor by the tumor cells. These models, indicating the
involvement of TGF-
in tumorigenesis, utilize heterologous
promoters; however, TGF-
gene transcription is also up-regulated
through its own promoter. For example, the TGF-
promoter regulates
expression of this gene through development (17) and mediates changes
induced by DNA methylation (18), hormones (19-23), and high glucose
concentrations (24). To determine how the endogenous TGF-
gene is
regulated, the promoters of the human and rat TGF-
genes have been
cloned (23, 25, 26). Their most characteristic features are the
apparent lack of TATA and CCAAT sequences (25) and high GC contents.
Elements in the human and rat TGF-
promoters have been partially
characterized. The human core promoter contains a nonconsensus TATA box
that is recognized by TATA-binding protein (27) and an initiator element that accurately orients the transcription complex, giving rise
to an unique transcription initiation site (28). In addition, Sp1
(human and rat) and p53 (human) play roles as transcriptional enhancers
for the TGF-
promoter (18, 27, 29).
In our characterization of the proximal TGF- promoter, we noted a
43-bp DNase I footprint that contained a GC-rich element that resembled
a consensus Sp1 binding site. This paper describes a more detailed
analysis of the proteins that bind to this element. These studies
revealed that the footprint results from the binding of two
sequence-specific transcription factors, one of which we have
identified as AP-2. These proteins bind simultaneously to this element
and are involved in the regulation of the 1.1-kb TGF-
promoter
activity. Our studies indicate that AP-2 regulates TGF-
transcription in vivo through its site in the TGF-
promoter. Our demonstration of the involvement of AP-2 in the
regulation of the TGF-
promoter may contribute to our understanding
of how TGF-
expression is regulated during carcinogenesis and during development.
The 220-bp TGF-
promoter (
181 to +38, relative to the transcription initiation site)
was cloned upstream of the luciferase gene in the pGL2 basic plasmid
(Promega) to make pGL-181/+38 as described previously (28). The 1.1-kb
TGF-
promoter (
1069 to +38) was cloned into the SacI
and HindIII sites in the same vector to give pGLT1.1.
Mutations were introduced into the TGF- promoter by site-directed
mutagenesis. pGL2 basic plasmid contains a bacteriophage replication
origin and was used to generate single-stranded template DNA. The
sequences of the mutagenic oligonucleotide primers for each mutant
promoter were as follows with the mismatch sequences underlined:
M
89/
96, 5
-GAGGACCGAGCGCCTAGAATTCAGCACTCGCCCCGCAG-3
; M
97/
103, 5
-GAGCGCCTCTCTGCTGAATTCTTCCCCGCAGCGCTGCC-3
;
M
104/
111, 5
-CTCTGCTGGCACTCGAGAATTCACGCTGCCCGCCGGGC-3
;
M
112/
118, 5
-GCACTCGCCCCGCAGTTGAATACGCCGGGCGGAAATAG-3
; M
121/
128,
5
-CCGCAGCGCTGCCCGTGAATTCTAAATAGGAAGGCGGC-3
.
T1.1PREm, T1.1 DREm, and T1.1(
104 m) contain the same mutation
as that of M
89/
96, M
121/
128, and M
104/
111, respectively,
and the same strategy was used to construct these mutants. The
introduction of mutations was verified by DNA sequencing.
To construct deletion mutant p89/
128, pM
89/
96, and
pM
121/
128 were digested with EcoRI. The resulting 5-kb
fragment from pM
121/
128 and the 0.7-kb fragment from pM
89/
96
were purified by agarose gel electrophoresis and ligated to each other
in proper orientation. To construct pD
68, the sequence from
65 to
70 in the TGF-
promoter was mutated to a ApaI site by
oligonucleotide-directed mutagenesis. The plasmid was then cut with
ApaI and EcoRI, and the 0.7-kb fragment was
isolated and ligated into the same sites in pBluescriptKS(+)
(Stratagene). The resulting plasmid was cut with HindIII and
KpnI, and the 110-bp fragment was ligated into the same
sites in pGL
181/+38, thus substituting for the wild type 220-bp
TGF-
promoter. The same strategy was used to construct pD
126
except that the sequence from
123 to
128 in the TGF-
promoter
was mutated to an ApaI site. pD
15 was constructed by digesting pGL
181/+38 with SacII and religating the
resultant ~5.8-kb fragment.
Oligonucleotides containing either wild type TRE I (
83 to
138 of
the TGF-
promoter) or T
RE I with a mutation at the distal regulatory element (DRE) were synthesized, with the HindIII
and XhoI sites at the 5
- and 3
-ends, respectively. These
oligonucleotides were annealed and ligated into the same sites in
pT81Luc (ATCC, plasmid 37584) to make pT
REI-TK and pDREm-TK. pSX is
a eukaryotic expression vector containing the SV-40 promoter. AP-2
cDNA was cloned into multiple cloning sites of pSX to make
pSX-AP2.
MDA468 and HepG2 cells were
maintained at 37 °C in a CO2 incubator in Dulbecco's
modified Eagle's medium supplemented with 10% newborn calf serum or
10% fetal bovine serum, respectively. Medium of HepG2 cells also
contains 0.1 mM minimum essential medium nonessential amino
acid solution (Life Technologies, Inc.). Cells were grown to
confluence, washed with phosphate-buffered saline, trypsinized, and
resuspended in full culture medium. For transfection with MDA468 cells,
approximately 1.0 × 107 cells were transfected by
electroporation with a total of 30 µg of plasmid DNA consisting of 20 µg of the indicated promoter-luciferase construct and 10 µg of the
pCMV--gal, a plasmid containing the CMV promoter-driving
-galactosidase as a control for transfection efficiency (18). For
transfection with HepG2 cells, approximately 5.0 × 106 cells were electroporated with 5 µg of the indicated
luciferase reporter plasmid, 5 µg of pCMV-
gal, and 1 µg of
either pSX or pSX-AP2. Cells were harvested 24 h after
transfection, and luciferase and
-galactosidase activities were
assayed as described previously (18).
Nuclear
extracts were prepared from either MDA468 cells or HepG2 cells (30).
Double-stranded oligonucleotides for gel mobility shift assays were
labeled with either [-32P]dATP or
[
-32P]dCTP using the Klenow fragment of DNA polymerase
I. The binding reactions were carried out at room temperature for 30 min in a total volume of 30-35 µl consisting of 50,000-80,000 cpm
of probes, 6 µg of MDA468 cell nuclear extract protein, 20 mM HEPES (pH 7.9), 25 mM KCl, 2 mM
spermidine, 0.1 mM EDTA, 2 mM
MgCl2, 10% glycerol, 1 mM dithiothreitol, 1 µg of poly(dI-dC), and 0.1 mg/ml bovine serum albumin, with or
without an excessive amount of competitor oligonucleotides. The
reactions were then resolved on a 6% nondenaturing acrylamide gel in
0.5 × Tris borate-EDTA buffer. For the antibody supershift assay,
0.5-2 ng of the rabbit polyclonal anti-AP-2 antibody (Santa Cruz
Biotechnology, Inc.) was also included in the reaction.
Oligonucleotides used in gel mobility shift assays were as follows
(only top strands are shown): DRE,
5-GATCTTCCTATTTCCGCCCGGCGGGCAGCGCTGAGACGC-3
; proximal regulatory
element (PRE), 5
-GCGCTGCGGGGCGAGTGCCAGCAGAGAGGCGCTC-3
; and
DREm,5
-GATCTTCCTATTTAGAATTCACGGGCAGCGCTGAGACGC-3
. Consensus oligonucleotides for AP-2 and Sp1 were obtained from Promega.
pGL-181/+38 was cut by
HindIII, and the exposed 3-end of the noncoding strand of
the TGF-
promoter was labeled with [
-32P]dATP using
the Klenow fragment of DNA polymerase I. The plasmid was then purified,
and the 220-bp radiolabeled fragment of the TGF-
promoter was
excised from the plasmid DNA using KpnI. The resulting
labeled fragment was purified by agarose gel electrophoresis. The
binding reaction for DNase I footprinting was carried out in a total
volume of 45 µl containing 30,000-50,000 cpm of probe DNA, 0 or 5 µl of partially purified MDA468 cell nuclear extract, 50 mM KCl, 0.1 mg/ml bovine serum albumin, 25 mM
Tris-HCl (pH 8.0), 6 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 100 ng of poly(dI-dC). After incubation on ice for 15 min, the
reaction was subjected to DNase I digestion in the presence of 2.5 mM CaCl2 for 1 min at room temperature. 100 µl of the stop solution (100 mM NaCl, 20 mM
EDTA, 1% SDS, 0.2 mg/ml proteinase K) was added to abolish DNase I
digestion. The DNA was then separated from the proteins by
phenol:chloroform extraction and ethanol precipitation and resolved on
a 5% denaturing polyacrylamide gel in a DNA sequencing gel apparatus
(Bio-Rad).
To identify the sequence(s) that is
important for TGF- promoter activity, 5
-deletion mutants of the
promoter were placed upstream of the luciferase gene and transfected
into MDA468 human mammary carcinoma cells. As shown in Fig.
1A, the deletion of the distal 1-kb sequence,
from
1069 to
181, caused only a minimal reduction (35%) of the
reporter activity. Strikingly, further deletion of a 114-bp fragment
(from
181 to
68) resulted in the almost complete loss of the
promoter activity. Similar results were obtained using HepG2 cells
(data not shown). These data indicate that this proximal region of the
TGF-
promoter contains most of the basal regulatory information for
TGF-
transcription.
To identify potential target(s) for transcription factors in this
proximal TGF- promoter, we performed DNase I footprinting experiments using the 220-bp promoter fragment (
181 to +38) as probe.
A 43-bp element was footprinted by MDA468 nuclear extract and
designated T
RE I (Fig. 1B, lanes 2 and
5). DNA sequences between
88 and
130 on the upper strand
and between
95 and
114 on the lower strand of the TGF-
promoter
were protected from DNase I digestion (Fig. 1C). TGF-
expression can be up-regulated by EGF (20-23). However, responsiveness
of the TGF-
promoter to EGF was not affected by the deletions shown
in Fig. 1A (data not shown). Also, protein binding to the
promoter remained unchanged upon EGF stimulation (compare lanes
2 and 3 and lanes 5 and 6, Fig.
1B). These data suggest that transcription factors acting through T
RE I regulate TGF-
basal transcription, whereas the EGF-responsive element resides elsewhere in the promoter.
To further define TRE I,
a series of site-directed mutations were introduced into this proximal
sequence (Fig. 2). The mutated TGF-
promoters were
cloned upstream of the firefly luciferase reporter gene and transfected
into MDA468 cells. Transcription from three of the mutant promoters,
M
89/
96, M
97/
103 and M
121/
128, was decreased to 38, 51, and
48% of the wild type promoter activity, respectively, whereas that of
M
104/
111 and M
112/
118, retained the wild type activity, at 120 and 97%, respectively. Again, T
RE I was found to be nonessential
for EGF stimulation because each mutant promoter responded equally well
to EGF with a 4-fold increase in reporter activity (data not
shown).
This mutational analysis suggests that TRE I contains two
cis-acting elements,
89 to
103 and
121 to
128,
designated the PRE and the DRE, respectively. Furthermore, in MDA468
cells, the T
RE I was able to enhance transcription from the
heterologous herpes simplex TK promoter, when placed upstream of this
promoter (Fig. 2). While mutations in either the PRE or DRE resulted in decreased promoter activity, simultaneous deletion of both the PRE and
DRE (
89/
128) resulted in the same loss of promoter activity that
was seen with mutation in each of the elements alone (Fig. 2). Whether
these elements act in concert in the regulation of the TGF-
promoter
activity such that both elements are necessary for full activity of the
T
RE I will be under further investigation. A similar transcription
requirement for T
RE I, especially the DRE, has also been observed in
HeLa cells (data not shown).
We determined whether protein binding to the TRE I was
affected by mutations at the PRE or DRE. We performed a DNase I
footprinting experiment on the 220-bp promoter fragments containing the
mutations at either the DRE or PRE (Fig. 3). We found
that the three mutant promoters, M
89/
96, M
97/
103, and
M
121/
128, which showed decreased promotor activities in the
transient transfection assays, also displayed altered patterns of
protein binding. Interestingly, mutations directed to either the PRE or
DRE alone only abolished protein binding to that same site but did not
affect protein binding to the other site. This result also indicates
that there are two protein binding sites in T
RE I that correspond to
the functionally defined DRE and PRE. The protein binding to either
site is independent on the protein binding to the other site.
T
To
further characterize the nuclear proteins that bind to the TRE I, we
used oligonucleotides containing either the PRE or DRE sequence as
probes in gel mobility shift assays (Fig. 4). Both PRE
and DRE oligonucleotides formed complexes with MDA468 nuclear proteins
(lanes 2 and 6). The complex formed on the DRE and PRE oligonucleotides had similar gel mobility, but the morphology of the bands were different. The DRE complex consisted of a relatively sharply defined band, whereas the PRE complex appeared more diffuse and
is of slightly higher mobility. The binding of these proteins to the
oligonucleotides was sequence-specific. The formation of these
complexes was inhibited by the presence of a 100-fold excess of the
corresponding unlabeled oligonucleotides (lanes 3 and
8) but the PRE and DRE oligonucleotides could not compete
with each other for protein binding (lanes 4 and
7). This observation suggests that different proteins bind
to these elements. We attempted to determine whether the PRE- and
DRE-binding proteins interact with each other with sufficient affinity
to allow the detection of a complex using the gel mobility shift assay.
Assuming that the PRE and DRE oligonucleotides are each bound by a
single protein (see below), interaction between these proteins could be
detected by the formation of a higher order and lower mobility complex in a binding reaction that included both oligonucleotides. However, in
the gel mobility shift assay using both oligonucleotides, we observed
only a simple mixture of the PRE and DRE complexes and no higher order
complexes (lane 9). As before, the competition reactions
confirmed the specificity of the binding proteins for their respective
elements (lanes 10 and 11). This result indicates that even if an interaction between these DNA-binding proteins can
occur, it is not sufficiently stable to allow detection by the gel
mobility shift assay.
AP-2 Binds to the DRE
Sequence analysis of the DRE indicated
the presence of a potential binding site for either transcription
factor Sp1 or AP-2 (Fig. 5A). In a gel
mobility shift assay using the DRE as a probe, a 100-fold excess of an
Sp1 consensus oligonucleotide failed to compete for protein binding
(Fig. 5B, left panel, lane 4).
However, a 50-fold excess of an AP-2 consensus oligonucleotides
completely inhibited the formation of the DRE-protein complex
(lane 5). Moreover, the mobility of the DRE-protein complex
was further retarded by the addition of rabbit polyclonal IgG against
AP-2 (lane 6), but not by the addition of
anti-retinoblastoma protein antibody (lane 7). In a parallel
experiment, a 25-fold excess of the DRE probe completely blocked
protein binding to the consensus AP-2 sequence (Fig. 5C,
lane 4). Taken together, these results indicate that AP-2
binds to the DRE. The complex formed on the PRE could not be
supershifted or inhibited by an AP-2 antibody (Fig. 5B,
right panel, lanes 3 and 4),
indicating that AP-2 neither interacts with the PRE nor is complexed by
protein-protein interaction with the transcription factor that binds
the PRE.
In Fig. 2, we have shown that a mutation at DRE decreased transcription
from the TGF- promoter to 48% of wild type (M
121/
128). An
oligonucleotide containing the same mutation failed to compete with
either the wild type DRE sequence or the consensus AP-2 element for
protein binding (Fig. 5C, lanes 7 and
11). Thus, the DRE is recognized in a sequence-specific
manner both in the intact cell and in vitro.
It has been previously
shown that the HepG2 human hepatoma cell line lacks endogenous AP-2
activity. Cotransfection experiments were done to test the functional
importance of the AP-2 site in the TGF- promoter (Fig.
6A). When the wild type T
RE I was placed upstream of a heterologous TK promoter (T
REI-TK), cotransfection of
a eukaryotic expression vector for AP-2 (pSX-AP2) increased transcription 2.2-fold compared with pSX alone. AP-2 cotransfection had
no significant effect on expression from the TK promoter alone. This
AP-2-dependent increase in transcription requires an intact DRE sequence, as indicated by the fact that AP-2 cotransfection failed
to activate when the T
RE I element contained the M
121/
128 mutation at the DRE site (DREm-TK).
That the transactivation by AP-2 is a result of AP-2 binding to the DRE
is further evidenced by a gel mobility shift assay using nuclear
extracts from HepG2 cells (Fig. 6B). No specific DNA/protein
interaction was detected on the DRE probe as indicated by competition
experiments using a 100-fold excess of the unlabeled probe (compare
lanes 2 and 3), consistent with the previous
observation that HepG2 cells have no endogenous AP-2 activity. However,
the addition of exogenous AP-2 protein resulted in three additional shifted bands, I, II, and III (lane 4). Complexes II and III
appear as strong shifts, whereas complex I is much weaker. A 100-fold excess of either unlabeled DRE or AP-2 DNA competed protein binding to
all three bands, indicating the specificity of these shifts (lanes 5 and 6, respectively). Complexes I, II,
and III were further confirmed to contain AP-2 by the quantitative
supershifting of these bands with -AP-2 antibody (lane
7).
The mutagenesis study in Fig. 2 used the proximal 220-bp
promoter. However, in vivo, TGF- transcription is
regulated in the context of a larger promoter. We therefore determined
the role of the DRE or PRE in the context of the entire promoter as it has thus far been defined. We constructed luciferase reporter constructs in which the DRE and PRE mutations were placed in the entire
1.1-kb (
1069 to +38) TGF-
promoter. Mutations in the DRE
(T1.1DREm) or PRE (T1.1PREm) that affected promoter function in the
context of the 220-bp proximal promoter had essentially the same effect
on the 1.1-kb promoter (Fig. 7), whereas a mutation between the PRE and DRE (T1.1(
104 m)) had no effect on transcription. This result further confirms the importance of these elements in
regulating the TGF-
gene transcription.
The transcription of the TGF- gene is regulated under a variety
of physiological and pathological circumstances. TGF-
is regulated
both temporally and spatially during development, resulting ultimately
in the expression of this growth factor in multiple tissues in the
adult organism. In most of the tissues examined, TGF-
expression
appears to be confined to specific cell types. For example, in the
bovine pituitary, maximal expression is seen in the lactotrophs (31);
in the ovary, the theca cells express TGF-
maximally just prior to
ovulation (32); in blood vessels, the arterial vascular smooth muscle
cells are the predominant site of expression (33); in the brain,
neurons in certain brain regions express TGF-
(34, 35), and in the
skin, the basal keratinocytes express this growth factor (21, 36).
Furthermore, TGF-
expression can be regulated in response to
metabolic and hormonal signals. In the mammary cells, estrogen (19,
37), phorbol esters (38), and EGF (or TGF-
itself) (20-22) can
regulate transcription of this gene. In vascular smooth muscle cells,
exposure to glucosamine or to superphysiological concentrations of
glucose (24) can up-regulate the transcription of this gene.
Interestingly, TGF-
expression was first observed in tumor cells
that had been transformed by retroviruses or spontaneously. The
expression in tumors was first detected because these transformed cells
express this growth factor at higher levels than the parental cell
lines from which they were derived. This observation of TGF-
overexpression in tumor cells gave rise to the autocrine hypothesis,
which was later confirmed by demonstrating that TGF-
overexpression
driven by heterologous promoters in cell lines (8-10) or in transgenic animals (11-14) results in the development of transformed cells or
tumors. That transcriptional activation of the TGF-
gene can lead to
marked phenotypic changes prompted us to investigate the endogenous
TGF-
promoter.
The TGF- promoter has many features in common with the promoters of
housekeeping genes in that it is GC-rich and does not contain a
consensus TATA-box. Like many housekeeping genes, transcription from
both the human (18) and rat (29) TGF-
promoters is highly dependent
upon the transcription factor, Sp1. The human TGF-
promoter also
contains an initiator element, which is required for optimal
transcription and appears to direct transcriptional initiation to a
unique site in the TGF-
gene (28). More recently, we have defined a
p53-response element in the human gene that may play a role in the
repair of epithelia suffering DNA damage from physical or chemical
agents (27). A nonconsensus TATA-box has also been defined in the human
TGF-
gene whose position and sequence is conserved in the rat gene
and that contributes to the transcriptional activity of the proximal
promoter (27).
In this paper, we describe another element in the proximal TGF-
promoter. This element, termed T
RE I, was first defined by DNase I
footprint analysis of the proximal promoter using nuclear proteins from
the human breast cancer cell line, MDA468. The T
RE I spans about 40 base pairs at a position centered around
110 relative to the
transcription initiation site. Deletion of the T
RE I reduces basal
TGF-
promoter activity, while this element activates transcription
from a heterologous TK promoter to 11-fold. Thus, T
RE I behaves like
a transcriptional enhancer in the TGF-
promoter. Further
characterization of the T
RE I indicated that it actually consists of
two elements that are recognized by distinct sequence-specific
transcription factors. We have termed these elements the DRE and PRE.
The evidence for the two elements consists of the following: (i) point
mutagenesis of the T
RE I defined two regions of this element that
contributed to the transcriptional enhancer activity both in the
context of the proximal promoter and the 1.1-kb promoter; (ii) the
point mutations that reduced enhancer activity reduced the span of the
DNase I footprint; (iii) gel mobility shift assays indicated that the
PRE and DRE were recognized by proteins having distinct DNA sequence
specificities; (iv) the DRE is recognized by AP-2. Although the DRE
contained a sequence resembling a binding site for Sp1, an AP-2 but not an Sp1 consensus oligonucleotide competed for binding to this segment
of the T
RE I, and the DRE is recognized by recombinant AP-2 when
added to the nuclear extract of HepG2 cells that are deficient in AP-2
activity. Also, the DNA-protein complex formed with the DRE was
recognized by an AP-2 antibody by not by a control antibody.
Furthermore, the DRE conferred sequence-specific AP-2 responsiveness to
a heterologous promoter when cotransfected into HepG2 cells with an
AP-2 expression vector. Although we have not identified the protein
that binds to the PRE, we do know that each protein can bind to its
portion of the T
RE I in the absence of the other as shown both in
the footprinting analysis and gel mobility shift assays. Furthermore,
AP-2 and the PRE-binding protein do not interact with each other with
sufficient affinity to allow detection by gel mobility shift assays.
Nevertheless, the DRE and PRE may cooperate functionally. Point
mutations in either the DRE or the PRE reduced the activity of the
T
RE I element as much as deletion of the entire T
RE I from the
promoter. The mechanism by which DRE and PRE may cooperate in
regulating the activity of T
RE I requires further investigation.
Although the AP-2 cDNA was cloned several years ago (39), the role
of this transcription factor in the control of gene expression remains
unclear. AP-2 is a 52-kDa protein that functions as a dimer,
recognizing a GC-rich DNA sequence 5-GCCNNNGGC-3
(39). Other similar
consensus binding sequences for AP-2 have also been reported. AP-2
binding sites are found in genes of SV40, human metallothionein IIa,
murine major histocompatibility complex H-2Kb, collagenase, human
growth hormone, human proenkephalin, human keratin K14, and mouse
mammary tumor virus (39-45). AP-2 appears to cooperate with other
transcription factors to mediate transcriptional activation in response
to signals induced by the developmental morphogen retinoic acid (39,
46) and to the signals mediated through the activation of
cAMP-dependent protein kinase and protein kinase C (41, 47,
48). In the case of the keratin K14 promoter, AP-2 appears to play a
role in the tissue-specific expression of K14 in keratinocytes, but it
does so in cooperation with a neighboring element (42). This
cooperative behavior might also exist between DRE and PRE of the
TGF-
promoter. AP-2 expression has been detected in the basal layers
of the skin (42, 49), and this correlation of AP-2, K14, and TGF-
localization in the skin is compatible with a role for AP-2 in the
control of the tissue-specific expression of these genes. AP-2 has also
been shown to play a role in the function of the oncoprotein, Ras. The
expression of AP-2 is stimulated by Ras transformation (50) in PA-1
cells. Interestingly, TGF-
expression is also increased in cells
transformed by Ras, suggesting a possible role for AP-2 in the Ras
induction of TGF-
(51). The finding of this element in the TGF-
promoter will contribute to our understanding of how the transcription
of this growth factor is regulated.
We thank Dr. Reinhard Buettner at the University of Regensburg (Regensburg, Germany), Dr. Trevor Williams at Yale University, and Dr. Gordon L. Hager at the National Institutes of Health for providing us the AP-2 cDNA and AP-2 cDNA under control of various promoters. We also thank the University of Alabama at Birmingham oligonucleotide core facility for synthesis of oligonucleotides. We are also grateful to the many helpful suggestions from members of the Kudlow laboratory.