(Received for publication, December 30, 1996, and in revised form, March 24, 1997)
From the Departments of Pathology,
Cell
Biology and Surgery, the ** Cell Adhesion and Matrix Research Center,
and the § University of Alabama Comprehensive Cancer Center,
University of Alabama at Birmingham, Birmingham, Alabama 35233-1924, and the ¶ Birmingham Veterans Affairs Medical Center,
Birmingham, Alabama 35233
Growth factors coordinately regulate a variety of
genes associated with pathological states including tumor invasion and
metastasis. Overexpressed epidermal growth factor receptor (EGFR) on
tumor cell surfaces is associated with enhanced cell attachment and migration into extracellular matrices, which promotes tumor
aggressiveness. We have demonstrated that epidermal growth factor (EGF)
up-regulates the cell surface adhesion molecule CD44 at both the
mRNA and protein levels on mouse fibroblasts expressing full-length
wild-type EGFR (NR6-WT) but not on EGFR-deficient cells (NR6-P). This
increases cell attachment to hyaluronic acid. In this investigation,
transcriptional regulation of CD44 by EGF was confirmed by defining an
EGF-regulatory element. By employing human CD44 gene
promoter-chloramphenicol acetyltransferase (CAT) constructs transfected
into NR6-WT cells, EGF inducibility was observed within a 120-base pair
(bp) DNA fragment located 450 bp upstream of the RNA initiation site.
Differential EGF inducibility was found among different cell lines
chosen, indicating a 3.2- and 1.8-fold enhancement in DU145 cells
carrying exogenous wild-type EGFR and in MCF-7 cells, respectively,
while minimal EGF induction was found in cervical cancer HeLa cells. Utilizing gel shift assays, a time-dependent increase of
DNA-protein complex formation was found upon EGF stimulation in NR6-WT
cells but not in NR6-P cells. Based upon these observations, a novel 22-bp EGF regulatory element (ERE)
(5-
604CCCTCTCTCCAGCTCCTCTCCC
583-3
)
was isolated from the CD44 gene promoter. This ERE conferred DNA-protein binding ability in vitro, as well as the full
functional recovery of EGF inducibility of CAT activity when linked to
a homologous CD44 promoter or a SV40 promoter driving a CAT reporter gene. A two-base mutation of the ERE completely eliminated its binding
activity as well as its EGF inducibility of CAT expression. Our studies
indicate that EGF induces CD44 gene expression through an interaction
between a specific ERE and putative novel transcriptional factor so as
to regulate cell attachment to extracellular matrix.
Growth factors in the extracellular milieu can convey signals to
cells via transmembrane surface receptors to regulate a variety of
downstream events. Activation of the epidermal growth factor receptor
(EGFR)1 upon binding of its ligands (EGF,
transforming growth factor-, HB-EGF, and amphiregulin) under both
normal and pathological conditions, initiates a cellular cascade which
results in major pleiotropic changes in many cell types (1, 2).
Aberrant overexpression of EGFR on tumor cells is associated with
increased proliferation, enhanced cell attachment, and migration into
extracellular matrices, progression to a transformed phenotype and
in vivo aggressiveness (3-7). Among the regulated genes are
those encoding cell surface adhesion molecules (8-10) as well as
nuclear transcriptional factors that regulate gene expression. Cell
surface glycoprotein CD44, an adhesion molecule that binds to the
extracellular matrix components, hyaluronic acid (11) and osteopontin
(12), has been postulated to modulate tumor invasion and metastasis
(13-15). Our previous studies with mouse 3T3 fibroblasts demonstrated
that EGF induced CD44 gene expression on both the protein and mRNA
levels and that this was accompanied by enhanced cell attachment to the
extracellular matrix component hyaluronic acid (16).
Although it is known that EGF regulates gene transcription of the early responsive genes, c-myc, c-fos, and c-jun, through signaling mechanisms that are shared with the protein kinase C signaling pathway (17-22), few EGF regulatory elements or their corresponding DNA-binding proteins have been identified. A serum-response element mediates EGF-stimulated c-fos transcription (23, 24). Sp1 is required by EGF to stimulate the human gastrin gene upon binding to a 16-bp overlapping GC-rich EGF response element (25), and elements in the prolactin and tyrosine hydroxylase promoter can serve as an EGF response element (ERE) (19, 23, 24, 26). However, these sequences do not connote a specific ERE.
Structural analysis of the CD44 upstream regulatory region in human
neuroblastoma cells (27) revealed that there were neither typical TATA
nor CCAAT boxes in the promoter region, although approximately 150 bp
adjacent to the 5-end of the gene itself was sufficient to induce
substantial basal transcription of the chloramphenicol
acetyltransferase (CAT) gene. Despite the existence of three consensus
hexanucleotide GGGCGG sequences, Sp1 binding to the CD44 gene upstream
region has not been demonstrated. Rather, the cis-regulatory
element, which was known to contribute to the down-regulation of CD44
in neuroblastoma cells, was identified as being a 120-bp DNA fragment
located 450 bp upstream of the RNA initiation site. To gain further
insight into the molecular mechanism by which EGF regulates CD44 gene
expression, we investigated transcriptional regulation of CD44 gene by
EGF using the bacterial CAT reporter system. Both in vitro
DNA-protein binding ability and in vivo EGF inducibility
were examined in both wild-type as well as mutational EREs. Herein, we
report the existence of a transferable 22-nucleotide sequence which
confers EGF responsiveness to transcriptional units.
Parental murine NR6 cells
(P), which are 3T3 derivatives devoid of endogenous EGFR (28), were
stably transduced via retrovirus-mediated infection with holo
(wild-type) EGFR (29). These cell lines have been previously
characterized, and are maintained at 37 °C, in a 5% CO2
environment, in minimal essential medium- (Life Technologies, Inc.)
supplemented with 7.5% fetal bovine serum, 100 µg/ml penicillin and
streptomycin, and 1 × non-essential amino acid and sodium pyruvate with 350 µg/ml G418 as a selective marker for the NR6-WT cells. Cells were carefully passaged before they reached 90%
confluence to eliminate spontaneous morphological change. EGF induction
was accomplished with 10 nM EGF (Life Technologies, Inc.)
in complete medium containing 0.1% dialyzed fetal bovine serum for
different time periods. Other cell lines used were: DU145 carrying
exogenous wild-type EGFR (31) in Dulbecco's modified Eagle's
medium/F-12 media supplemented with 100 µg/ml penicillin and
streptomycin, 7.5% fetal bovine serum, and 1 mg/ml G418 as a selective
marker; MCF-7 and HeLa cells were maintained according to
recommendations from the ATCC.
Preparation of nuclei and RNA labeling followed standard
protocols (30). Briefly, 1 × 108 NR6-P or NR6-WT
cells were treated with 10 nM EGF for 0, 1, 3, 6, or
12 h, washed with ice-cold phosphate-buffered saline, and preincubated in cold lysis buffer (10 mM Tris-Cl (pH 7.4),
3 mM CaCl2, and 2 mM
MgCl2). The cells were resuspended in 2 ml of lysis buffer
followed by further lysis in 2 ml of Nonidet P-40 buffer (addition of
1% Nonidet P-40 to the lysis buffer) before disruption by 10 strokes
in a cold Dounce homogenizer. Disruption was confirmed by light
microscopic evaluation after trypan blue staining. Nuclei were pelleted
by centrifugation at 500 × g, at 4 °C for 5 min,
and then resuspended in ice-cold glycerol storage buffer (50 mM Tris-Cl (pH 8.3), 40% glycerol, 5 mM
MgCl2, and 0.1 mM EDTA) followed by immediate
return to dry ice before being stored at 70 °C for later use.
Elongation of nascent RNA from 5 × 107 nuclei was
carried out for 30 min at 30 °C in 200 µl of 2 × reaction
buffer (10 mM Tris-Cl (pH 8.0), 5 mM
MgCl2, 0.3 M KCl, 3 mM
dithiothreitol, 35 units of RNasin, and 1 mM each of CTP,
ATP, and GTP) in the presence of 10 µl of [-32P]UTP
(760 Ci/mmol, 10 mCi/ml, Amersham). After treatment with 24 µg of
DNase I (Sigma) for 15 min at 30 °C and 10 µg of proteinase K for
30 min at 42 °C, labeled RNA was extracted with phenol/chloroform and precipitated with 5% trichloroacetic acid. Precipitants were then
applied to Whatman GF/A glass fibers which were further washed 3 times
with 10 ml of 5% trichloroacetic acid, 30 mM sodium
pyrophosphate and incubated in 1.5 ml of reaction buffer (20 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM CaCl2) in the presence of 40 µg of DNase I
for 30 min at 37 °C. RNA was eluted by heating the mixture to
65 °C for 10 min after adding 45 µl of 0.5 M EDTA and
68 µl of 20% SDS and then treating with 10 µg of proteinase K at
37 °C for 30 min, before precipitating with 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol. Aliquots of
~4-5 × 106 cpm of labeled RNA in 1 ml of TES
solution (10 mM TES (pH 7.4), 10 mM EDTA, and
0.2% SDS) were hybridized to nitrocellulose membranes previously
dot-blotted with 5 µg of denatured and linealized plasmid DNA
containing CD44 cDNA,
-actinin cDNA, or vector DNA in the presence of 1 ml of TES/NaCl solution (0.6 M NaCl + TES
solution) at 65 °C for 36 h. After washing twice in 25 ml of
2 × SSC for 1 h at 65 °C followed by 8 µg of RNase A
treatment at 37 °C for 30 min, nitrocellulose strips were then
unraveled and exposed to x-ray film.
Plasmids containing different fragments of the human CD44
gene regulatory region placed upstream of the bacterial CAT gene (pCATbasic, Stratagene) (27) were a kind gift from Dr. Emma Shtivelman
(SYSTEMIX, CA). Specifically, these plasmids were: pRBCAT with 1.9 kilobases of genomic CD44 sequences extending upstream from the
BamHI site in the first exon; pSacBCAT and pRVBCAT were 5
truncated versions of pRBCAT, with 0.65- and 0.53-kilobase inserts
upstream of the CAT gene, respectively; and pRB-SRCAT which was a
deletion version of the pRBCAT sequence in which a 120-bp fragment
between the SacI and EcoRV sites were excluded (illustrated in Fig. 2).
To further define the putative ERE, three repeats (22 bp,
5-CCCTCTCTCCAGCTCCTCTCCC-3
) were amplified by polymerase chain reaction methods from a synthesized oligonucleotide template
(5
-ACCCAAGCTTCGCCCTCTCTCCAGCTCCTCTCCCGCATGCGTCGACCGCCCTCTCTCCAGCTCCTCTCCCGCTAGCCGCCTTCTCTCCAGCTCCTCTCCCGTCGACGAT-3
) using the 5
primer (5
-ACCCAAGCTTCGCCCT-3
) and the 3
primer (5
-ATCGTCGACGGGAGAG-3
), and placed upstream of the pRVBCAT plasmid as
p3ERE-RVBCAT. p3ERE-RVBCAT then underwent internal restriction enzyme
digestion with SphI and SalI followed by
religation to establish a p2ERE-RVBCAT and p1ERE-RVBCAT which contained
2 or 1 ERE repeats, respectively. A heterologous SV40-driven CAT
reporter system was also utilized to verify the ERE function.
Synthesized oligonucleotides of the ERE as
5
-pGATCTAAGCTTGGGAGAGGAGCTGGAGAGAGGGCGATATCA-3
and
5
-p-GATCTGATATCGCCCTCTCTCCAGCTCCTCTCCCAAGCTTA-3
were
annealed and ligated into a BglII site in a pPromoterCAT
vector (Promega) to obtain both a sense oriented
(pERE(s)SV40CAT) and antisense oriented
(pERE(as)SV40CAT) CAT construct. Two-base mutations of the
ERE (from T to C) synthesized from oligonucleotides as
5
-CCCTCTCCCCAGCTCCCCTCCC-3
and its
compliment, were placed in front of an SV40 promoter of the
pPromoterCAT vector at a Klenow-blunted BglII site to
establish a heterologous ERE mutation construct
(pERE(Mu)SV40CAT), after polymerase chain reaction
screening and DNA sequencing confirmation.
DNA transfection was performed on 1 × 107 of 80%
confluent NR6-P and NR6-WT cells using equimolar amounts of pCAT
constructs corresponding to 20 µg of pCAT-basic DNA (Promega) by an
electroporation method (Bio-Rad) under constant conditions (400 V, 500 microfarads capacity, and 0 resistance). Plasmids containing a
-galactosidase gene driven by an SV40 promoter were used to
normalize the transfection efficiency for basal levels of CAT
expression. In the EGF induction CAT assays, transfected cells were
equally divided, allowed to recover in complete medium for 24 h,
and then treated with or without 10 nM EGF for an
additional 24 h in 0.1% dialyzed fetal bovine serum containing
medium. CAT activity was measured from cell extracts by the CAT Enzyme
Assay System (Promega) based on liquid scintillation counting using
14C-labeled chloramphenicol (Amersham) and
n-butynyl coenzyme A. The level of CAT activity from pRVBCAT
transfectants without EGF induction was set at 100% and the percent
(fold) increase in the other transfectants determined and compared with
the standard. Five independent experiments, each in duplicate, were
performed for each plasmid transfection. Statistical significance was
determined by use of the Student's t test.
5 × 107 cells
were lysed, and nuclei collected as above, following 10 nM
EGF induction of both NR6-P and NR6-WT cells for 0, 1, 3, 6, or 12 h. Disruption of the nuclear membranes was accomplished by sequentially
adding 1/2 packed nuclear volume of a low salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM
MgCl2, 60 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5 mM
dithiothreitol) and then an equal volume of a (1/2 packed nuclear volume) high salt buffer (1.2 M KCl as 60 mM in
low salt buffer). The solution was gently mixed for 30 min before
centrifugation at 14,500 rpm for 30 min. Supernatants were tube
dialyzed (6,000-8,000 Mr cut-off) against a buffer
containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol at
4 °C for 2 h followed by centrifugation to remove precipitated
protein and nucleic acids. Supernatants containing nuclear extracts
were further purified by heparin-affinity chromatography (Bio-Rad) and
eluted from a concentration gradient of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 M KCl in TM buffer (25 mM Tris-Cl (pH
8.0), 6 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 15% glycerol). Aliquots that contained the most significant binding to the DNA probe were stored at
70 °C and utilized in the subsequent gel shift and DNA
footprinting assays. The final concentration of extracts was estimated
by both a Bio-Rad protein assay and quantitative gel shift assay.
HindIII-digested pSacBCAT plasmid was Klenow
end-labeled with [-32P]dCTP before a second digestion
with EcoRV. The released 120-bp labeled DNA fragment was
fractionated and purified on agarose gel. Complementary synthetic
oligonucleotides of putative ERE sequence (Fig. 6) were annealed before
T4 kinase 5
-end-labeling with [
-32P]ATP. 2 × 104 cpm of each probe was applied to 20 µl of premixed
gel shift reaction containing 25 mM Tris-Cl (pH 8.0), 6.25 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and 40 ng of
poly(dI·dC) in the presence of 4 µg of nuclear extract at 25 °C
for 1 h. Poly(dI·dC) at 0.2 or 1 µg, unlabeled 120-bp DNA
probe at 1 or 100 ng, and unlabeled pRVBCAT insert DNA at 1 or 100 ng
were preincubated with the nuclear extracts at 25 °C for 10 min
before being placed in reaction with the labeled probe which served as
the competition control. Annealed synthetic oligonucleotides were
5
-end-labeled with
-32P by T4 kinase before being used
in gel shift reactions. DNA-protein complexes were resolved on a native
4% nondenatured polyacrylamide gel containing 45 mM Tris
base, 45 mM boric acid, and 2.5% glycerol under
nondenaturing conditions. After exposure to x-ray film, gel shift bands
were quantified by densitometry (Image 1.47 software).
DNase I Protection Footprinting Assays
DNase I protection
assays were performed in 50 µl of reaction with 0.5, 2, or 4 µg of
nuclear extract from NR6-WT cells treated with or without 10 nM EGF for 6 h and 2 × 104 cpm of
the 120-bp [-32P]dCTP-labeled DNA probe as described
in the gel shift assay. The nuclear extract was preincubated without
probe for 10 min on ice in the same buffer as used in the gel shift
assay, and incubated for another 30 min on ice after the addition of
probe. Digestion was initiated with the addition of 1 unit of DNase I (Promega) in 25 mM CaCl2 at room temperature
and terminated after 1 min with 90 µl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml
yeast RNA) prior to phenol-chloroform extraction and ethanol
precipitation. Digested DNA and sequencing reactions of the probe using
sequencing primer, 5
-AGCTTCTCCCTCTTTCCAC-3
carried out by the
Sequenase version 2.0 (U. S. Biochemical Corp.), was resolved on a 5%
polyacrylamide, 7 M urea gel followed by autoradiography.
Our previous data
indicated that EGF up-regulated cell attachment of NR6 mouse
fibroblastic cells to hyaluronic acid by increasing the expression of
the cell surface adhesion molecule CD44 at both the protein and
mRNA levels (16). EGF treatment of NR6 cells expressing wild-type
EGFR resulted in a 3-4-fold up-regulation of CD44 protein expression
with a maximum at 24 h, as well as a 3-fold increase of mRNA
levels after 6-24 h of EGF treatment (16). The CD44 mRNA in NR6
cells were determined by nuclear run-on analysis to confirm the
transcriptional regulatory mechanism (Fig. 1). Both NR6
parental cells without endogenous EGFR (P) and NR6 cells with
transfected EGFR (
WT) were preincubated with 10 nM EGF
for various times before collecting and labeling new transcripts. In
agreement with earlier Northern blot analysis, there was no perceptible
change in the rate of CD44 transcription with NR6-P cells, while a
3-fold induction was observed maximally at 6 h in NR6-WT cells in
the presence of EGF. This was seen to decline by a third at 12 h,
identical to the transient wave of mRNA seen on Northern blot
analysis (data not shown). These results support the contention that
EGF regulates CD44 mRNA levels by increasing transcription in a
time-dependent manner at least during short-term EGF
exposure.
Functional Characterization of the EGF Regulatory Element (ERE) in the CD44 Gene
To identify the sequence that mediates EGF responsiveness in the CD44 gene, a series of DNA fragments containing the sequence of the human CD44 gene upstream regulatory region (pRBCAT) and restriction enzymatic internal truncations (pSacBCAT and pRVBCAT) and deletions of the human CD44 gene upstream region (pRB-SRCAT) were obtained and placed into a bacterial CAT reporter gene (Fig. 2A). CAT activities were measured on electroporated transfected NR6-WT cells in the presence or absence of EGF. Consistent with the previous finding in human neuroblastoma cells (26), all constructs that contained the 150-bp sequence upstream of the transcription initiation site conferred substantial basal CAT activity on the constructs (Fig. 2B) when compared with the promoterless CAT plasmid.
The observation that not only did pSacBCAT elicit a 2-fold higher basal CAT activity than pRVBCAT or pRB-SRCAT, both of which were lacking a 120-bp fragment between SacI and EcoRV sites (Sac-RV fragment), but that the same order of magnitude change was seen in comparison to pRBCAT, which contains this fragment, suggested that a negative cis-element upstream of the SacI site and a stimulatory cis-element on the Sac-RV fragment may contribute to the basal promoter activity. Moreover, as there was an increase in EGF-mediated induction of CAT activity in pRBCAT and pSacBCAT (2.5- and 2.3-fold, respectively) (Fig. 2C), the only construct with a Sac-RV fragment sequence, this was strong presumptive evidence that an ERE was present in this region.
Differential EGF Induction of CAT Gene Transcription between Different CellsTo test whether the observed relatively low EGF
induction in NR6 cells (2.3-fold) was due to low promoter activity or
is the result of a cell-specific effect, three transformed human cell lines, which express EGFR and CD44 and are derived from prostate, DU145-WT, breast, MCF-7, or cervix HeLa, were employed in our transfection/CAT system (Fig. 3). All cells were
transfected by pCAT, pRVBCAT, and pSacBCAT constructs via standard
electroporation methods. Basal level of CAT activity was determined by
cotransfection of -galactosidase and was shown to be similar among
these cells (data not shown). After EGF stimulation for 24 h
following transfection, changes in CAT activity was determined by
comparing EGF-treated cells with their non-EGF-treated counterparts. In
agreement with the data from NR6 cells, no significant CAT induction
was found following mock (pCAT basic vector) and pRVBCAT transfections
in all cell lines tested; however, a differentiated CAT induction by
EGF stimulation was demonstrated in these cells, being 2.3-, 3.3-, 1.8-, and 1.2-fold in NR6-WT, DU145-WT, MCF-7, and HeLa cells,
respectively. The extent of chloramphenicol acetylation did not
increase in the presence of a 4-fold excess of n-butynyl coenzyme A; thus, this substrate was not limiting under the conditions of the assay. Moreover, additional experiments demonstrated that CAT
induction of NR6-WT cells began at a low dose of 5 nM EGF and was sustained at an EGF concentration of up to 20 nM
(data not shown), indicating no EGF dose-dependent
bidirectional effect.
A EGF Inducible Nuclear Protein Binds to the Sac-RV Fragment of the CD44 Promoter
The presence of an EGF inducible DNA binding
activity was sought by an electrophoretic mobility shift assay using
the 32P-labeled Sac-RV DNA fragment (which contained the
putative ERE described in the functional analysis), and nuclear
extracts from NR6-P and -WT cells. Pilot experiments indicated that
nuclear extracts eluted from heparin-affinity columns with 0.4 M KCl buffer contained the DNA-protein complexes of
interest. Binding specificity was assessed by examining the effect of
excess amounts of poly(dI·dC) as nonspecific DNA, unlabeled Sac-RV
fragment as specific competitor, and probe containing the CD44 promoter
sequence from the pRVBCAT plasmid as random nonspecific DNA (Fig.
4). It was noted that three bands (labeled 2, 3, and 4) were eliminated by preincubating the labeled
probe with 0.5 µg of poly(dI·dC), while the major band (labeled
1) retained significant binding, even in the presence of
excess poly(dI·dC) (Fig. 4A). Furthermore, the major band
could be competed only with increasing amounts of specific unlabeled Sac-RV probe (10-100 ng, equivalent to 20-200-fold excess of the labeled probe) (Fig. 4B) but not to excessive nonspecific
probe derived from pRVBCAT (Fig. 4C).
The time course of EGF induction of this ERE response was assessed
using nuclear extracts from both NR6-P and -WT cells, treated by EGF
for 1, 3, 6, or 12 h (Fig. 5). Extracts were
normalized to cell number with the loading amount ranging from 3.8 to
4.0 µg; 0.5 µg of poly(dI·dC) (500-fold excess) was also included in the reaction mixture to minimize nonspecific binding. No change was
observed in protein-DNA complex formation upon EGF stimulation in the
NR6-P cells. However, in NR6-WT cells, the induction became evident
(2-fold increase, n = 3), 3-6 h after exposure to EGF, and persisted for at least 12 h (Fig. 5B). It was noted
also that this time-dependent induction of protein-DNA
complex formation paralleled the mRNA wave demonstrated in both
Northern blot and nuclear run-on assays, suggesting that the induction
response required functional EGFR expressed on cell surfaces and that
the Sac-RV fragment contained an ERE.
Identification of an ERE in the CD44 Gene Promoter
This work
strengthened the hypothesis that EGF up-regulates CD44 gene expression
through induction of transcriptional factor(s) binding to an ERE in the
gene promoter region. To determine the specific sequence to which the
transcriptional factor bound, we performed DNase I footprinting by
using a nuclear extract from NR6-WT cells induced by EGF for 6 h
and [-32P]dCTP 5
-end (Klenow) labeled Sac-RV as the
DNA probe. Two "footprints" were observed when 0.5 µg of
poly(dI·dC), as nonspecific DNA competitor, was included in the
binding reaction: footprint I over the sequence
5
-CTCCTCTCCC
583 and footprint II over the sequence
-
604CCCTCTCTCC
595 (Fig. 6).
Both sites were better protected from DNase I digestion when an
increasing amount of nuclear extract (0.5, 2.0, and 4.0 µg in
lanes 2, 3, and 4, respectively, from non-EGF
treated cells; lanes 5, 6, and 7, respectively,
from EGF-treated cells) was added. The specificity of the protein
binding was also demonstrated by competition with excess poly(dI·dC),
as nonspecific DNA, which abolished a putative third footprint and with
unlabeled Sac-RV fragment, as specific probe, which competed away all
footprints while the RVB fragment, acting as an unlabeled random
nonspecific competitor, did not affect the DNase I protection (data not
shown). It should be noted that both footprints are partially
palindromic in themselves and are inverse repeats of each other, and
that the 2-base nucleotides between them were partially protected. A
sequence search in GenBank against known transcriptional factors did
not reveal any significant homology to the two separate footprints, either alone or together. Therefore, this sequence from
583 to
604
on the CD44 gene upstream regulatory region appears to be a novel
EGF-regulatory element in mouse fibroblast NR6 cells.
The
sequence-specific binding of the NR6 cell nuclear extract was further
studied in a gel shift assay by using the
32P-5-end-labeled Sac-RV fragment and an unlabeled 22-bp
double-strand synthetic oligonucleotide as competitor:
5
-CCCTCTCTCCAGCTCCTCTCCC-. The protein-DNA complex formation
recognized as the EGF-inducible nuclear protein was noted to decrease
as an increasing amount of unlabeled synthetic oligonucleotides (1-10
pmol, equivalent to 10- and 100-fold excess of labeled probe,
respectively) was applied to the gel shift reaction mixture (Fig.
7B). This suggested that this synthetic
sequence could competitively bind the same EGF-induced protein.
To confirm the binding specificity of this ERE, the Sac-RV fragment
(probe 1), the 22-bp oligonucleotide sequence (probe 2), and two
oligonucleotides containing either the 5 or 3
half of the palindromic
structure (5
-CCCTCTCTCCAG- and 5
-CAGCTCCTCTCCC-) (probes 3 and 4, respectively) were 5
-end-labeled with [
-32P]ATP
followed by incubation with the NR6-WT nuclear extract and a gel shift
assay was performed. As shown in Fig. 7A, only the 22-bp ERE
(probe 2), but not the half-sequences (probe 3 and 4), was capable of
binding the band compatible with the Sac-RV probe, indicating that
EGF-induced protein-DNA binding requires a full-length sequence.
The time-dependent EGF responsiveness was further demonstrated by using nuclear extracts from EGF-treated NR6-WT cells and labeled ERE (Fig. 7C). EGF inducibility was evident from 3 to 6 h after EGF stimulation, similar to the findings using the labeled Sac-RV probe. Together, these results further strengthen the conclusion that the 22-bp sequence is an EGF-regulatory element which regulates CD44 gene expression.
ERE Is Necessary for EGF-induced TranscriptionWe
demonstrated that the ERE sequence within the CD44 gene promoter is
critical for both EGF-induced DNA-protein complex formation and
EGF-induced transcription of the CAT gene. The identity of the ERE in
the context of EGF induction of CD44 gene transcription was further
assessed in a homologous and a heterologous CAT reporter system.
Multiple EREs were placed in front of the CD44 gene promoter in the
pRVBCAT construct, which lacked EGF inducibility of CAT gene
transcription as described above. They were p1ERE.RVBCAT, p2ERE.RVBCAT, and p3ERE.RVBCAT containing 1, 2, and 3 ERE,
respectively. pRVBCAT and pSacBCAT served as the negative and positive
controls. After transfection of NR6-WT cells with the various
constructs, half the cell groups were stimulated with 10 nM
EGF for 24 h and compared in the CAT assay to the untreated
matched control for increase of CAT activity secondary to EGF
induction. All ERE-containing constructs elicit similar significant
induction of CAT activity by EGF (2.8-, 2.4-, and 2.5-fold for
p1ERE.RVBCAT, p2ERE.RVBCAT, and p3ERE.RVBCAT, respectively), similar to
the positive control (2.3) fold (Fig. 8A).
The fact that p1ERE.RVBCAT yielded at least equal EGF induction may
imply that accessibility of the ERE because of its relative shorter
sequence may be important in this process, although other mechanisms
may also account for the observed changes.
To rule out the possibility that the CAT induction by EGF was the result of transcription initiated from the transcription start site of the transfected CD44-CAT reporter gene, the ERE sequence was introduced into a heterologous SV40 promoter-driven CAT reporter system in both a sense and antisense orientation (pERE(s)SV40CAT and pERE(as)SV40CAT, respectively). Both constructs and the pSV40CAT vector control conferred high constitutive basal level of CAT activity (data not shown). However, only the sense ERE sequence was able to fully restore the EGF induction of CAT transcription in NR6-WT cells (2.3-fold increase), while minimal (1.2-fold) to no induction was observed in pERE(as)SV40CAT and pSV40CAT vector, respectively.
The Integrity of the 22-bp ERE of CD44 Promoter Is Necessary for Full Transcriptional InductionEGF-induced CD44 transcription is
initiated by a specific nuclear protein binding to the ERE sequence of
the CD44 gene promoter. We performed an experiment to test the
specificity of the ERE sequence in the EGF induction event by point
mutating two "T" nucleotides to two "C" (Fig.
9C), to disturb the potential binding motif
(palindromic structure). The binding activity to the nuclear protein
was measured in a gel shift assay. EGF inducibility of CAT
transcription was also verified in a pSV40CAT reporter
system by placing the mutated ERE in front of the SV40
promoter. In this analysis, the specific DNA-complex formation was
observed to be enhanced when an unmutated ERE (WT) and an increasing
amount of nuclear extract from EGF-treated NR6-WT were placed in the
gel shift reaction mixture. Minor bands were, however, noted above the
specific bands and were thought to be the result of nonspecific binding
since no increasing binding effect was found when increasing amounts of
nuclear extract was applied. Poly(dI·dC), a nonspecific competitor,
was able to eliminate these bands without affecting the major specific
DNA-protein complex formation. In contrast, no significant DNA-protein
complex formation was detected using the mutated ERE even in the
presence of an excess amount of nuclear extracts (10 µg) (Fig.
9A). More importantly, transfection of the wild-type ERE
(WT)-SV40CAT reporter resulted in a full restoration of EGF
induction of CAT activity (2.3-fold increase) in NR6-WT cells, while
the same reporter with a mutated ERE (Mu) devoid of DNA-protein binding
capacity exhibited no induction (1.0-fold) (Fig. 9B).
Although inducible activity was relatively low in this experiment, the
results clearly establish the necessity for the integrity of this ERE
sequence for EGF induction.
EGF is known to regulate many different gene transcripts and thereby modulate cell adhesion, attachment, migration, and differentiation, under both physiologic and pathologic conditions (31). We have earlier shown that EGF up-regulates the cell surface adhesion molecule CD44 protein and its mRNA level to enhance mouse fibroblast NR6 cell attachment to the extracellular matrices (16). In the present study, using different techniques, we have confirmed that EGF induces CD44 gene transcription and that this induction is mediated by a novel EGF regulatory element found in the upstream regulatory region of the CD44 gene.
The ERE sequence differs from previously reported EGF response elements found in genes encoding c-myc, c-fos, c-jun, and other genes whose protein products include gastrin (31), prolactin, tyrosine hydroxylase (19, 23, 25, 26), transin (20), and pS2 (21). These studies have shown that EGF stimulates transcription of genes through different cis-regulatory sequences. The ERE of c-fos is mediated by a serum response element (32) and the Sp1 site (GGGCGG) modulates the gastrin gene, whereas EGF induction of tyrosine hydroxylase and prolactin is mediated by the nearly identical sequence GGAAGAGGATGCC (19, 23). Although an ERE sequence containing the sequence GGGCGG has been identified as a high affinity Sp1 site (33), Sp1 is unlikely to mediate CD44 transcriptional activation by EGF in our model system because the pRVBCAT plasmid, which contains this sequence, did not show EGF induction. Moreover, gel shift assays using labeled probe containing the Sp1 sequence, released from the pRVBCAT construct, did not demonstrate significant binding to nuclear extracts of NR6 cells (data not shown). As noted above, to exclude other possible transcriptional factors which may bind to this ERE, we searched data bank files and confirmed that the CD44 gene's ERE is not similar to other described regulatory elements.
EGF induction of CAT activity in our ERE sequence is relatively low
(~2.3-fold). This most likely is due to low intrinsic promoter
activity, similar to that found in other gene promoters, i.e. the -fibroblast growth factor regulatory element of
the skeletal
-actin promoter (34). This also suggests that EGF may
modulate cell attachment to the extracellular matrices via the
coordination of multiple cell surface adhesion molecules (8-10) rather
than exclusively by CD44. However, the possibility of cell-specific differential induction could not be completely ruled out because in our
transfection system a 2.3-, 3.2-, and 1.8-fold induction of CAT
activity was detected in mouse fibroblasts, human prostate cancer, and
human breast cancer cells, respectively.
Unlike other cis-acting EGF regulatory elements that may
also mediate transcriptional activation by phorbol esters (35-37), we
have preliminary data that other growth factors including transforming growth factor- and basic fibroblast growth factor, but not phorbol 12-myristate 13-acetate, induced CD44 protein levels in NR6 cells. This
would suggest that EGF stimulates CD44 transcription through a distinct
receptor signaling event other than the protein kinase C signaling
pathway. Whether these growth factors activate the same transcription
element or different factors which bind to unrelated
cis-regulatory sequences needs to be addressed. Like the
serum response element-mediated c-fos transcription, our ERE binds to nuclear proteins from EGFR-devoid NR6-P cells and unstimulated NR6-WT cells so as to activate basal transcription in the context of
the CD44 promoter when linked to CAT reporter gene. The specific DNA-protein complex formation derived from NR6-WT cells increased after
EGF stimulation in a time-dependent manner. This implies that stimulation of CD44 gene transcription by EGF results from either
an increased expression or increased activation of this cis-acting DNA-binding protein.
We noted that the 22-bp ERE sequence contains the palindrome of
5-CCCTCTC- and -CTCTCCC-3
. Although the significance of this is
currently unknown, it might serve as a DNA recognition site for either
multimeric proteins composed of identical subunits or is needed to form
hairpin loops to generate structures that might be differentially
recognized by specific DNA-binding proteins. As demonstrated in the gel
shift assay, each half of the ERE sequence was unable to form a stable
DNA-protein complex. It is most likely that binding of nuclear protein
to each CCCTCTC sequence of the CD44 ERE is required for protein-DNA
recognition. Cloning of the gene encoding the ERE-binding protein will
permit further characterization of its structure and the process by
which EGF exerts a stimulatory effect on CD44 gene expression.
In summary, we conclude that EGF transcriptionally up-regulates CD44 gene expression. The 22-bp ERE from the CD44 gene promoter region is orientation-specific, and is necessary to enable EGF-induced CD44 gene transcription through a specific interaction between the ERE motif and a putative novel transcriptional factor.
We thank Dr. Emma Shtivelman (SYSTEMIX, CA) for the kind gift of the cloned CD44 promoter constructs. Furthermore, we thank Victor Krasnykh and Dr. David Curiel, Gene Therapy Program, University of Alabama at Birmingham, for assistance in the oligonucleotide synthesis.