From the Department of Pharmacology and Vincent T. Lombardi Cancer Center, Georgetown University, Washington, D. C. 20007
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ABSTRACT |
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Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element.
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INTRODUCTION |
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FGF-BP1 is a secreted protein that binds to acidic FGF and basic FGF in a non-covalent reversible manner (1). FGF-BP mRNA has been found to be up-regulated in squamous cell carcinoma (SCC) cell lines of different origin, in SCC tumor samples from the head and neck, and in some colon cancer cell lines (1, 2). More recently, developmental expression of the mouse FGF-BP gene was found to be prominent in the skin and intestine during the perinatal phase and is down-regulated in adult mice (3). We previously described that expression of FGF-BP in a non-tumorigenic human cell line (SW-13) which expresses bFGF leads to a tumorigenic and angiogenic phenotype (2). Expression of FGF-BP in these cells solubilizes their endogenous bFGF from its extracellular storage and allows it to reach its receptor, suggesting that FGF-BP serves as an extracellular carrier molecule for bFGF (2, 4). Expression of FGF-BP under the control of a tetracycline-responsive promoter system in SW-13 cells revealed its role during the early phase of tumor growth (5). To assess the significance of FGF-BP endogenously expressed in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their FGF-BP by targeting with specific ribozymes (6). This study showed that the reduction of FGF-BP reduced the release of biologically active bFGF from cells in culture. In addition, the growth and angiogenesis of xenografted tumors in mice was decreased in parallel with the reduction of FGF-BP, suggesting that some human tumors can utilize FGF-BP as an angiogenic switch molecule.
The fact that FGF-BP has been detected in only a few types of tumors, where it seems to play a crucial role in angiogenesis, led us to investigate the mechanisms responsible for turning its expression on or off. Studying the regulation of FGF-BP in SCC cell lines, we showed that all-trans-retinoic acid, used as a chemotherapeutic agent against SCCs, down-regulates FGF-BP gene expression in vitro by both transcriptional and post-transcriptional mechanisms (7). In vivo all-trans-retinoic acid treatment reduces FGF-BP expression in SCC xenografts and inhibits their tumor growth and angiogenesis (8). On the other hand, FGF-BP mRNA expression in the adult mouse skin was found to be dramatically increased during the early stages of 7,12-dimethylbenz[a]anthracene/TPA-induced mouse skin papilloma formation (3), as well as in 7,12-dimethylbenz[a]anthracene/TPA-treated human skin grafted onto SCID mice.2 Similarly, FGF-BP expression in vitro was up-regulated in epidermal cell lines carrying an activated ras gene, implicating the ras/PKC pathway in the regulation of FGF-BP (3).
In this context, and given the fact that FGF-BP could play a critical role in the development of human skin cancer, we decided to investigate the effects of the tumor promoter TPA on FGF-BP gene regulation. Our results show that FGF-BP mRNA expression is up-regulated by TPA in the ME-180 SCC cell line and that this induction is mediated by direct transcriptional mechanisms. Analysis of the human FGF-BP promoter reveals that the TPA induction is mediated by cooperation of several inducible regulatory elements. Furthermore, the induction of gene expression by TPA can be modified by a repressor element juxtaposed to the AP-1 site which contains sequences recognized by E box element factors.
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MATERIALS AND METHODS |
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Cell Culture-- The ME-180 squamous cell carcinoma cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in improved minimum essential medium (Biofluids Inc., Rockville, MD) with 10% fetal bovine serum (Life Technologies, Inc.).
Northern Analysis--
ME-180 cells were grown to 80%
confluence on 150-mm tissue culture dishes, washed three times in
serum-free IMEM, and then treated 16 h later with
12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma) in
serum-free IMEM. Total RNA was isolated with the RNA STAT-60 method
using commercially available reagents and protocols (RNA
STAT-60TM, Tel-Test, Friendswood, TX). 30 µg of total RNA
were separated by electrophoresis in 1.2% formaldehyde-agarose gel and
then blotted onto nylon membranes (MSI, Westboro, MA). The blots were
prehybridized in 6× SSC (0.9 M sodium chloride, 0.09 M sodium citrate, pH 7.0), 0.5% (w/v) SDS, 5× Denhardt's
solution (0.1% (w/v) Ficoll, 0.1% (w/v) polyvinylpyrrolidone, 0.1%
(w/v) bovine serum albumin, 100 µg/ml sonicated salmon sperm DNA)
(Life Technologies, Inc.) for 4 h at 42 °C. Hybridization was
carried out overnight at 42 °C in the same buffer. After
hybridization, blots were washed three times with 2× SSC and 0.1% SDS
for 10 min at 42 °C and finally once with 1× SSC and 0.1% SDS for
20 min at 65 °C. Autoradiography was performed using intensifying
screens at 70 °C. Blots were stripped by boiling 2 × for 10 min in 1× SSC and 0.1% SDS. Hybridization probes were prepared by
random-primed DNA labeling (Amersham Pharmacia Biotech) of purified
insert fragments from human FGF-BP (2) and human GAPDH
(CLONTECH). The final concentration of the labeled probes was always greater than 106 cpm/ml hybridization
solution. Quantitation of mRNA levels was performed using a
PhosphorImager (Molecular Dynamics).
In Vitro Transcription on Isolated Nuclei--
ME-180 cells were
grown to 80% confluence on 150-mm tissue culture dishes. Cells were
washed three times in serum-free IMEM and then treated 16 h later
with TPA in serum-free IMEM for indicated times. Nuclei from
107 cells for each time point were isolated after
incubation in lysis buffer containing 0.5% Nonidet P-40 as described
(7). Nuclear transcription assays were performed with
[-32P]UTP (Amersham Pharmacia Biotech) as described
(7). Equal amounts of radioactivity (0.5-1 × 107
cpm) were hybridized to nitrocellulose filters containing 3 µg of
each plasmid. After hybridization for 4 days at 42 °C, the filters
were washed 4 times with 2X SSPE, 0.1% SDS for 5 min at 25 °C and
treated for 30 min at 25 °C in 2X SSPE containing 20 µg/ml RNase
A. The filters were then washed 4 times for 30 min in 1X SSPE, 1% SDS
at 65 °C. The amount of radioactivity present in each slot was
determined using a PhosphorImager after overnight exposure, and
autoradiograms were exposed for 1-3 days with intensifying screens.
Primer Extension--
Primer 1 was designed from the coding
region of the human FGF-BP cDNA (5'-GTGAGGCTACAGATCTTC-3'), primer
2 from the FGF-BP 5'-UTR (5'-GTTCACCTTGTTCTGAGCACACGGATCCA-3'), and a
control primer for the 1.2-kb kanamycin RNA (Promega). 10 pmol of each
primer was labeled with T4 polynucleotide kinase (Promega) and 30 µCi of [-32P]ATP (Amersham Pharmacia Biotech) for 1 h, and labeled primers were purified over a ChromaSpin-10 gel
filtration column (CLONTECH). Total RNA from ME-180
cells was isolated as described for Northern analysis. ME-180 mRNA
was purified from total RNA over an oligo(dT)-cellulose column (Life
Technologies, Inc). 100 fmol of each FGF-BP-specific primer was
incubated with or without 7 µg of ME-180 mRNA or control primer
with or without 2 ng of 1.2-kb kanamycin control RNA (Promega) in the
presence of avian myeloblastosis virus reverse transcriptase buffer (50 mM Tris-HCl, 8 mM MgCl2, 30 mM KCl, 1 mM DTT, pH 8.5, Boehringer Mannheim)
and allowed to anneal for 1 h at 50 °C. Annealed mixtures were
then incubated in the presence of 2 mM dNTPs, 50 units of
RNase inhibitor (Boehringer Mannheim), and 40 units of avian
myeloblastosis virus reverse transcriptase (Boehringer Mannheim) for
1 h at 42 °C. Samples were then run on a 6% polyacrylamide sequencing gel along with radiolabeled HinfI markers and
exposed overnight for autoradiography.
Cloning of FGF-BP Gene Promoter-- 1.8 kb of genomic sequence lying upstream of the human FGF-BP gene was isolated from a human genomic library using the PCR-based PromoterFinder DNA Walking Kit (CLONTECH) according to the manufacturer's recommendations using rTth XL DNA polymerase (Perkin-Elmer). Gene-specific primers derived from the 5'-UTR of the human FGF-BP cDNA were 5'-ACACGGATCCAGTGCAATCC-3' (+91 to +72) for the primary round of PCR and 5'-GGAGTGAATTGCAGGCTGCAGCTGTGTCAG-3' (+62 to +33) for secondary PCR. Secondary PCR products from a DraI library (1.1 kb) and PvuII library (1.8 kb) were cloned into a TA Cloning Vector pCR2.1 (Invitrogen), sequenced by automated cycle sequencing (ABI PRISM Dye Terminator Cycle Sequencing, Perkin-Elmer), and confirmed to contain contiguous genomic sequence. This sequence has been submitted to GenBankTM (accession number AF062639).
Plasmid Constructs--
Promoter fragments were cloned into the
PXP1 promoterless luciferase reporter vector (9). PCR products from
PvuII (1829/+62) and DraI (
1060/+62)
libraries were removed from pCR2.1 using HindIII and
XhoI sites in the pCR2.1 multiple cloning site and ligated
into the HindIII and XhoI site of PXP1 to
generate pX-1829/+62Luc and pX-1060/+62Luc. Both constructs contained a
BamHI site carried over from the pCR2.1 vector and located
3' of the FGF-BP sequence and 5' of the luciferase gene. The 5'
promoter deletion constructs pX-118/+62Luc, pX-93/+62Luc, pX-77/+62Luc,
pX-67/+62Luc, pX-56/+62Luc, and pX-31/+62Luc were generated by PCR from
pX-1060/+62 Luc template using upstream primers spanning
118 to
100,
93 to
76,
77 to
59,
67 to
49,
56 to
35, and
31
to
11, respectively, all containing a 5'-linked BamHI
site. Downstream primer was derived from the luciferase gene
5'-CCATCCTCTAGAGGATAGAATGGCGCCGGGCC-3'. PCR was carried out using
Taq DNA Polymerase (Boehringer Mannheim). Products were cut
with BamHI, gel-purified, and cloned into the PXP1
BamHI site. Correct sequence and orientation were verified by dideoxynucleotide chain termination sequencing with a Sequenase kit
2.0 (U.S. Biochemical).
Transient Transfections and Reporter Gene Assays--
24 h
before transfection, ME-180 cells were plated in 6-well plates in IMEM,
10% FBS at a density of 750,000 cells/well. For each transfection, 1.0 µg FGF-BP-luciferase construct and 10 µl of LipofectAMINE Reagent
(Life Technologies, Inc.) were combined in 200 µl of IMEM, and
liposome-DNA complexes were allowed to form at room temperature for 30 min. Volume was increased to 1 ml with IMEM, added to rinsed cells, and
incubated for 3 h at 37 °C. Cells were washed and incubated in
IMEM for 3 h and then treated for 18 h with vehicle alone
(Me2SO, final concentration 0.1%) or 107
M TPA. Transfection efficiency for each construct was
determined by co-transfection with 1.0 ng of a CMV-driven
Renilla luciferase reporter vector pRL-CMV (Promega) and
found to be the same for all BP-PXP1 constructs. However, due to a
2-fold background TPA induction of pRL-CMV (see above), results were
normalized for protein content and not for Renilla
luciferase activity. Cells were lysed by scraping into 150 µl of
Passive Lysis buffer (Promega), and cell debris was removed by brief
centrifugation. 20 µl of extract was assayed for both firefly and
Renilla luciferase activity using the
Dual-LuciferaseTM Reporter assay system (Promega). Light
intensity was measured in a Monolight 2010 luminometer. Light units are
expressed firefly light units/µg of protein. Protein content of cell
extracts was determined by Bradford assay (Bio-Rad).
Gel Shift Assays--
ME-180 cells were grown to 80% confluency
on 150-mm dishes, serum-starved for 6 h, and treated with or
without 107 M TPA for 90 min. Nuclear
extracts were prepared according to Dignam et al. (11) with
the following modifications. Pelleted cells were resuspended in 1 ml of
buffer A (15 mM KCl, 10 mM HEPES, pH 7.6, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, 1 mM sodium
orthovanadate) (12) with 1× CompleteTM protease inhibitor
mixture (Boehringer Mannheim) and incubated on ice for 10 min. Crude
nuclei were pelleted at 700 g and resuspended in 50 µl of
ice-cold buffer C (0.42 M NaCl, 20 mM HEPES, pH
7.9, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 1 mM sodium orthovanadate, 1× CompleteTM
protease inhibitor mixture) and vortexed at 4 °C for 15 min. After
centrifugation for 10 min at 1000 × g, supernatant was
used directly in binding assays and stored at
70 °C.
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RESULTS |
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TPA Increases FGF-BP mRNA in SCCs--
We have previously
detected an up-regulation of FGF-BP mRNA following TPA treatment of
mouse skin during the development of skin tumors (3) and also in human
skin xenografts.2 These data suggest that the control of
this angiogenic switch factor may play an important role in skin
carcinogenesis. To examine this further we studied the effect of the
tumor promoter TPA on FGF-BP gene expression in ME-180 cells which
express high levels of the FGF-BP transcript (7). Cells were treated
with 107 M TPA from 1 to 24 h which
resulted in an increase in the steady-state levels of FGF-BP mRNA
detectable 1 h after treatment (Fig.
1A). PhosphorImager analysis
showed that the induction was maximal after 6 h by 452 ± 44% (Fig. 1A). GAPDH mRNA remained unaffected by TPA
treatment, as judged relative to the total amount of RNA loaded and was
used to standardize FGF-BP mRNA. The dose dependence of TPA
induction of FGF-BP mRNA in ME-180 is shown in Fig. 1B. We estimated the half-maximal effective concentration as 1 nM. The inductive effect of TPA on FGF-BP mRNA was also
observed in two other SCC cell lines, FaDu and A431 (data not shown)
demonstrating that TPA induction of FGF-BP mRNA is generally
preserved in SCC cell lines.
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Mechanism of TPA Induction of FGF-BP mRNA--
We have
previously shown that FGF-BP gene expression can be regulated through
both transcriptional and post-transcriptional mechanisms (7). Therefore
we next attempted to determine whether the TPA induction of FGF-BP
mRNA was at the transcriptional or post-transcriptional level. We
first assessed whether TPA treatment affected the stability of the
FGF-BP mRNA. Experiments were performed to determine whether
addition of inhibitors of transcription (actinomycin D) or translation
(cycloheximide) could inhibit the TPA induction of FGF-BP mRNA.
Actinomycin D (5 µg/ml) or cycloheximide (10 µg/ml) were added with
or without TPA (107 M), and FGF-BP mRNA
levels were determined 6 h after treatment. As shown in Fig.
2A, simultaneous addition of
TPA and actinomycin D completely blocked the TPA induction, whereas
simultaneous addition of TPA and cycloheximide had no effect. These
data suggest that TPA directly increased the rate of FGF-BP gene
transcription independently of de novo protein synthesis and
did not affect the stability of the FGF-BP transcript. To verify
further that the stability of the FGF-BP transcript was not modified by
TPA treatment, ME-180 cells were pretreated for 2 h with TPA and
then actinomycin D was added to inhibit transcription. As shown in Fig.
2B, pretreatment of cells with TPA did not increase the
half-life of the FGF-BP mRNA indicating that the stability of the
FGF-BP transcript is not affected by TPA.
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Isolation and Characterization of the Human FGF-BP Promoter-- In order to understand better the transcriptional regulation of the human FGF-BP gene, 1.8 kb of genomic sequence upstream to the known 5'-UTR sequence of human FGF-BP cDNA was isolated from a human genomic library and sequenced. The transcription start site of the human gene was determined using primer extension analysis with nested primers derived from known cDNA sequence (Fig. 4). The precise start site compatible with the primer extension results is indicated in Fig. 5. Alignment between the human and mouse FGF-BP promoter which we cloned previously (3) revealed a region of high homology with 70% nucleotide identity within the first 200 nucleotides upstream from the transcription start (Fig. 5). Nucleotide homology dropped significantly in more upstream sequences, suggesting that the proximal conserved 200 nucleotides of the promoter could be important for transcriptional regulation of FGF-BP in both species.
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Functional Analysis of the Human FGF-BP Promoter--
To identify
the functional promoter elements involved in FGF-BP gene regulation by
TPA, progressive 5' deletion mutants were constructed based on the
location of consensus factor binding sites on the promoter. Deletion
constructs were transiently transfected into ME-180 cells, and their
relative luciferase activity was assayed in the absence or presence of
TPA (Fig. 6). The basal activity of each
vector is shown in the left panel of Figs. 6 and 7. The
empty PXP1 vector had no detectable luciferase activity either in the
absence or presence of TPA (data not shown). However, we did observe a
background, approximately 2-fold, TPA induction of the PXP1 vector when
several unrelated minimal promoters (i.e. thymidine kinase
minimal promoter, the CMV minimal promoter, and the
pro-opiomelanocortin minimal promoter) were treated with TPA after
transfection into ME-180 cells (Fig. 6, control vector). Background induction by TPA of a variety of vectors has been described previously and is presumably mediated through cryptic sites in the PXP1
plasmid (20). The TPA induction due to the inserted FGF-BP promoter was
considered to be that observed above the background control vector
induction. The FGF-BP promoter from 1060 to +62 was induced about
4-fold above control vector in the presence of TPA and showed the same
TPA inducibility as the full-length 1.8-kb promoter construct (data not
shown). Deletion from
1060 to
118, which removed 950 base pairs of
promoter sequence including the potential NF-
B site, had no effect
on TPA induction and was also induced 5-fold above background (Fig. 6).
Similarly, deletion from
118 to
93, which removed one of the
potential Ets-binding sites, retained full TPA induction. Removal of
the consensus Sp1 binding site from
93 to
77 had no effect on TPA
induction of the FGF-BP promoter. However, the 5' deletion to
77
caused an 80% decrease in basal activity of the promoter (Fig. 6,
left panel) suggesting that the Sp1 consensus site is a
predominant mediator of basal promoter activity of FGF-BP but is not
required for TPA induction.
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Contribution of Ets, AP-1, and C/EBP Sites to TPA
Induction--
In order to better understand the contribution of each
individual consensus binding site to TPA induction, internal promoter deletions were introduced and tested for TPA inducibility within the
context of the promoter from
118 to +62. Deletion of the Ets site
alone (
76 to
67) or deletion of the AP-1 site alone (
65 to
58)
reduced TPA induction slightly to the intact promoter (Fig.
7). Deletion of both Ets and AP-1 (
76
to
58), however, resulted in a significant decrease in both basal
activity and in TPA induction, indicating that both sites act in
cooperation for full promoter activity. However, loss of the juxtaposed
Ets/AP-1 site does not completely abolish TPA induction, suggesting
that additional sites are also involved.
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TPA Regulation of the FGF-BP Promoter Involves a Repressor Element
Juxtaposed to the AP-1 Site--
Between the AP-1 site and the
C/EBP site lies a region of low homology between the human and mouse
EGF-BP promoter sequences. Because this region was not suspected to
have any effect on TPA induction, an internal deletion removing this
region (
57 to
47) was tested as a control. Surprisingly, in the
57/47 construct, TPA induction of the FGF-BP promoter increased from
approximately 5 to 11-fold, suggesting the presence of a possible
repressor which may interact with this site. The lack of sequence
conservation between the human and mouse in this region may reflect a
difference in the regulation of FGF-BP between the two species. The
57 to
47 deletion disrupts an AACGTG (
60 to
55) which is
juxtaposed to the 3' end of the AP-1 site and which shows some
similarity to the CACGTG E box element recognized by a number of
helix-loop-helix factors (24). To test this imperfect E box for
repressor activity, a C to T point mutation at position
58 was
introduced into the
118/+62 BP promoter construct (Fig. 7). This
mutant-58 (m-58) construct showed a dramatic increase in TPA induction
up to 16-fold above background. Moreover, when the
58 point mutation
is introduced into the
C/EBP
construct (Fig. 7,
C/EBP
/m-58), this promoter mutant also showed
increased fold induction by TPA, suggesting that repression mediated by
this site is not dependent on the C/EBP
site. These data show that
the point mutation at position
58, as well as the internal deletion
from
57 to
47, disrupts repression of the FGF-BP promoter which
normally limits the response to TPA.
Transcription Factor Binding to FGF-BP Promoter Elements--
In
order to ascertain that TPA induction of FGF-BP was due to direct
activation by transcription factors, we performed gel retardation
analysis to show transcription factor binding to FGF-BP promoter
elements. By using labeled promoter sequence from 80 to
63
containing the putative Ets-binding site as a probe (Fig. 8A), the binding of three
specific protein complexes in the presence of ME-180 nuclear extracts
was detected (Fig. 8B). Protein binding to all three
complexes was increased in the presence of TPA (Fig. 8B, lane
3) and was specifically competed away in the presence of excess
unlabeled
80/
63 oligonucleotides (lanes 4 and
5). Further competition analysis showed that the factors
binding to the
80/
63 element were only weakly competed by consensus
Ets elements from the collagenase promoter (25) and polyoma virus enhancer (26), requiring over 100-fold excess in order to compete for
binding (data not shown). It has previously been described that
specific residues flanking the GGA trinucleotide motif of the Ets site
are required for high affinity sequence-specific binding of individual
Ets family members (27-29). Therefore, our data suggest that the
80/
63 element on the FGF-BP promoter could bind an Ets family
member other than the Ets-1 or Ets-2 proto-oncogenes (26).
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DISCUSSION |
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In this report we demonstrate that TPA induction of FGF-BP
mRNA levels is primarily through stimulation of gene transcription. This is in contrast to the retinoid repression of FGF-BP gene expression which we have previously shown is mediated through post-transcriptional and transcriptional mechanisms (7). In fact, at
least at early time points after retinoid administration, the post-
transcriptional mechanism which is dependent on new protein synthesis
predominates since the half-life of the FGF-BP mRNA is greater than
16 h (7). Our studies show that the TPA induction of FGF-BP
mRNA is rapid, requiring no new protein synthesis and involves
direct activation by transcription factors whose site of action is
clustered in the first 118 base pairs upstream of the transcription
start site. Within this region the majority of the TPA stimulation of
the FGF-BP promoter can be explained by the additive effects of two
sites positioned between 76 to
58 and from
47 to
33.
The 76 to
58 site harbors a perfect consensus to the AP-1
transcription factor binding site NTGAGTCA (31). The AP-1 transcription factor complex comprises the c-fos and c-jun
proto-oncogenes which are known to be activated as a result of TPA
stimulation of PKC-dependent pathways (32). However,
deletion of the AP-1 site alone in the FGF-BP promoter caused only a
slight reduction in TPA effects on the FGF-BP promoter. This result is
consistent with the emerging picture that AP-1 acts synergistically
with other transcription factors, such as the Ets family of
transcription factors, to mediate gene expression in response to TPA
and other stimuli (28, 29). In the FGF-BP promoter deletion of
sequences 5' to the AP-1 consensus significantly decreases the TPA
stimulation in comparison with deletion of the AP-1 site alone. These
5' sequences contain the core GGA found in the center of the Ets family
DNA consensus recognition site (29). Considering the body of evidence
that suggests that Ets/AP-1 cooperate for full transcriptional
activation, it seems likely that this may be the function of the
76/
58 element. For instance similar cooperation between Ets and
AP-1 occurs through a juxtaposed Ets/AP-1-binding site in the polyoma
virus enhancer (18) and has subsequently been implicated in the
regulation of genes involved in invasion and metastasis, including
collagenase and urokinase plasminogen activator (19, 33-37). Although
we found that the collagenase Ets element or the polyoma virus Ets element did not effectively compete for binding to the FGF-BP Ets
element, this may reflect the binding of another Ets family member to
the FGF-BP promoter whose recognition site could be determined by
sequences flanking the GGA core (27).
Deletion of the 47 to
33 FGF-BP promoter region also substantially
reduces the TPA effects on the FGF-BP promoter. Sequence analysis
revealed that a site homologous to the C/EBP
-binding site is
centered in this region of the promoter. The factors binding to FGF-BP
C/EBP
element, however, are not effectively competed by the C/EBP
site from the p21WAF1/CIP1
gene promoter, suggesting that transcription of FGF-BP may be mediated
by a different C/EBP
-related factor. The published consensus for
C/EBP
is T(T/G)NNGNAA(T/G) (38) which is identical in
eight positions (underlined) to the site between
48 to
41 differing only in the most 3'-nucleotide of the consensus. In addition, the
involvement of C/EBP
in TPA-mediated responses has been shown previously. For instance, induction by phorbol esters has been shown to
cause increased C/EBP
synthesis, phosphorylation, and DNA binding to
promoters of a number of genes including MDR1 and collagenase 1 (21-23, 39). Thus, C/EBP
or a family member is involved in the
activity of the
47 to
33 element. Like other leucine zipper family
members, C/EBP
acts cooperatively with other transcription factors
to modulate the level of gene expression in response to extracellular
stimuli. For example, C/EBP
has been shown to associate with Fos/Jun
in vitro (40) and can cooperate in vivo to induce
expression of the TSG-6 gene in response to interleukin-1
and tumor necrosis factor-
which is mediated through distinct AP-1
and C/EBP
-binding sites in the TSG-6 promoter (41). Similarly, our data show that the C/EBP
consensus element is a major
mediator of TPA-induced gene expression of FGF-BP. However, because
removal of the C/EBP
site alone does not completely abolish TPA
induction, this suggests that like other TPA-induced genes, the
C/EBP
site acts in cooperation with other promoter elements.
A novel aspect of TPA regulation of the FGF-BP promoter is the role of
the region 57 to
47 between the AP-1 site and the C/EBP
site.
Deletion of this region substantially increases the TPA response,
implying that this region normally represses the extent of the response
to TPA. A point mutation in this region also abrogates repression thus
making it unlikely that the effect of the deletion is simply to bring
the AP-1 and C/EBP
sites in closer proximity leading to their
increased responsiveness to TPA. In fact, the relief of repression
obtained with the
58 point mutant is observed in the presence of the
C/EBP
deletion suggesting that the repression impacts on the AP-1
element rather than the C/EBP
site. An alternate possibility is that
the factor bound to the
57 to
47 site interacts with the general
transcription machinery in a manner similar to the NC2 repressor (42).
However, this seems less likely because we observe no increase in basal activity of the promoter after deletion or mutation of the repressor site in comparison to the
118 construct (Fig. 7). The
57 to
47
deletion destroys an AACGTG (
60 to
55) which is a variant of the
CACGTG E box element recognized by a number of helix-loop-helix factors
(24). The
58 mutant changes the AACGTG to AATGTG and would perturb
the 5' part of the dimer recognition sequence (24). However, the wild
type sequence alone does not predict which member of the
helix-loop-helix family would interact with this site. Interestingly,
binding to an AACGTG recognition element has been described in
vitro to a homodimer of the aryl hydrocarbon receptor nuclear
translocator helix-loop-helix factor (43), and aryl hydrocarbon
receptor nuclear translocator-deficient embryonic stem cells have a
defective angiogenesis process (44). However, it is unclear whether
aryl hydrocarbon receptor nuclear translocator homodimers interact with
promoters in vivo. Alternatively, other helix-loop-helix
factors are known to function as transcriptional repressors, such as
the Mad family of proteins that bind related E box sequences during
TPA-induced macrophage differentiation (45, 46) and recruit the
mSin3-histone deacetylase corepressor complex, leading to a more closed
chromatin structure and transcriptional repression (47).
Through gel retardation analysis, we show distinct factor binding to the AP-1 site and to the E box repressor site. Interestingly, factor binding to both of these sites is increased upon stimulation with TPA. TPA-induced transcription factor binding to E box elements has been described for a number of different promoters including c-fos (48-50). The observation that TPA induces factors which both stimulate and limit induction of FGF-BP suggests a mechanism by which transcription of the FGF-BP gene could be tightly regulated and may reflect a level of tissue-specific expression of this gene.
Overall, our data suggest that the TPA induction of the FGF-BP promoter
is induced through both Ets/AP-1 site and a C/EBP site and that the
extent of induction is moderated by factors that bind to an E box
repressor element which lies adjacent to the AP-1 site. It is known
that TPA also induces the expression of genes involved in proteolytic
degradation of the extracellular matrix such as stromelysin,
collagenase, and urokinase plasminogen activator (33, 51, 52).
Interestingly, these promoters are regulated by similar transcription
factors as those which we show are involved in FGF-BP promoter
induction, e.g. Ets/AP-1 and C/EBP
. Thus, our data would
support the argument that a specific subset of transcription factors
may be induced (or derepressed) to specifically stimulate a panel of
genes involved in invasion, angiogenesis, and metastasis during skin
tumor development.
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ACKNOWLEDGEMENTS |
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We thank Achim Aigner and Rafael Cabal-Manzano for sharing unpublished data and all members of the Riegel and Wellstein labs for lively discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK02141 (to A. T. R.) and CA71508 (to A. W.), a U. S. Army Medical Research and Material Command pre-doctoral fellowship (to V. K. H.), a Susan Komen Foundation award (to A. W.), and American Cancer Society Grant CB 82807 (to A. W.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF062639.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: The Research Bldg., E307, Georgetown University, 3970 Reservoir Rd., N.W., Washington, D. C. 20007. Tel.: 202-687-1479; Fax: 202-687-4821.
1 The abbreviations used are: FGF-BP, fibroblast growth factor-binding protein; bFGF, basic FGF; TPA, 12-O-tetradecanoylphorbol-13-acetate; SCC, squamous cell carcinoma; PKC, protein kinase C; C/EBP, CAATT/enhancer binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IMEM, improved minimum essential medium; kb, kilobase pair(s); DTT, dithiothreitol; PCR, polymerase chain reaction; UTR, untranslated region; CMV, cytomegalovirus.
2 A. Aigner and A. Wellstein, unpublished data.
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