J Biol Chem, Vol. 275, Issue 15, 10943-10953, April 14, 2000
E-cadherin Is a WT1 Target Gene*
Seiyu
Hosono
,
Isabelle
Gross§,
Milton A.
English,
Karen
M.
Hajra¶,
Eric R.
Fearon¶, and
Jonathan D.
Licht
**
From the Derald H. Ruttenberg Cancer Center,
Department of Medicine, and ** Department of Biochemistry and
Molecular Biology, Mount Sinai School of Medicine, New York, New York
10029 and the ¶ Department of Internal Medicine, Division of
Molecular Medicine & Genetics, University of Michigan Medical Center,
Ann Arbor, Michigan 48109
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ABSTRACT |
The WT1 tumor suppressor gene encodes a
transcription factor that can activate and repress gene expression.
Transcriptional targets relevant for the growth suppression functions
of WT1 are poorly understood. We found that mesenchymal NIH 3T3
fibroblasts stably expressing WT1 exhibit growth suppression and
features of epithelial differentiation including up-regulation of
E-cadherin mRNA. Acute expression of WT1 in NIH 3T3 fibroblasts
after retroviral infection induced murine E-cadherin expression. In
transient transfection experiments, the human and murine E-cadherin
promoters were activated by co-expression of WT1. E-cadherin promoter
activity was increased in cells overexpressing WT1 and was blocked by a
dominant negative form of WT1. WT1 activated the murine E-cadherin
promoter through a conserved GC-rich sequence similar to an EGR-1
binding site as well as through a CAAT box sequence. WT1 produced
in vitro or derived from nuclear extracts bound to the
WT1-response element within the murine E-cadherin promoter, but not the
CAAT box. E-cadherin, a gene important in epithelial differentiation
and neoplastic transformation, represents a downstream target gene that
links the roles of the WT1 in differentiation and growth control.
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INTRODUCTION |
The WT1 tumor suppressor gene is associated with Wilms' tumor,
which can be considered a disease of disordered development, since the
tumors exhibit the three elements of primordial kidney development
including blastemal, stromal, and epithelial elements (1). The WT1 gene
is predominantly expressed in the developing kidney, but WT1 expression
is also detected in the fetal gonad, spleen, mesothelium, breast, and
other tissues (reviewed in Ref. 2). During early nephrogenesis, WT1
expression is first detected in the metanephric mesenchyme. At the
onset of epithelial differentiation, WT1 expression increases as the
mesenchyme surrounding the ureteric bud condenses to form primary
vesicles. These cells further differentiate into comma- and S-shaped
bodies, and subsequently mature into glomeruli and proximal tubules.
This phenomenon and the finding that mice null for WT1 fail to develop
kidneys (3) suggest an essential role for WT1 in controlling the
mesenchymal-epithelial transition of renal development. WT1 differs
from other tumor suppressor genes such as Rb and p53 in that Rb and p53
are widely expressed in a variety of developing and adult tissues,
while WT1 expression is restricted. The restricted pattern of WT1
expression suggests that its growth-suppressive properties may be
linked to its role in organogenesis.
WT1 gene encodes a Cys-His zinc finger transcription factor, which
functions as both a transcriptional repressor and activator (2, 4-6).
The DNA binding activity of WT1 was demonstrated on several different
sequences including 5'-GCGGGGGCG-3', which is also
recognized by EGR-1, EGR-2, and EGR-3 (4, 7-10). Alternative splicing
of the WT1 gene yields four major isoforms of the WT1 protein. Isoforms C and D include a 3-amino acid (KTS) segment between
zinc fingers three and four of the protein, which alters the DNA
binding specificity of the protein, while isoforms B and D include a
17-amino acid segment involved in transcriptional regulation. WT1(A)
includes neither additional segment (2).
The tumor suppressor activity of WT1 was demonstrated by stable
transfection of wild-type WT1 into a Wilms' tumor cell line harboring
a mutant WT1 (11), as well as in heterologous osteosarcoma, CV1, and
fibroblast cells (12-15). WT1 can regulate expression of a number of
growth and differentiation-related genes in co-transfection assays
including insulin-like growth factor II (16, 17), insulin-like growth
factor I receptor (18), platelet-derived growth factor A-chain (19,
20), Pax-2 (21), syndecan-1 (22), Dax-1 (23), and
amphiregulin (24) (reviewed in Refs. 2 and 25). Epidermal growth factor
receptor down-regulation by WT1 may contribute to growth suppression by
inducing apoptosis (12), while syndecan activation (22) and
amphiregulin (24) secretion may be important for renal differentiation.
A problem in the interpretation of many of these studies has been the
lack of a model for the action of WT1 in inducing both differentiation
and growth control. We found that expression of WT1 in mesenchymal
fibroblasts both suppresses cell growth (13) and induces features of
epithelial differentiation (26). These WT1-expressing NIH 3T3 cells
exhibit desmosome-like structures and increased expression of several
epithelial marker genes such as E-cadherin, type IV collagen, and
perlecan (heparin sulfate proteoglycan) along with reduced expression
of 
8 integrin and vimentin (26). Furthermore, we
recently identified, by representational difference analysis, genes
up-regulated in WT1-expressing cell lines which are expressed in the
condensing metanephric mesenchyme of the developing mouse
kidney.1 This lends support
to our hypothesis that expression of WT1 in this heterologous system
mimics some of the effects of WT1 in kidney differentiation. Similarly,
work by Haber's group identified amphiregulin as a gene induced by WT1
in an osteosarcoma cell line but also expressed in the condensing,
developing human kidney (24). In this study we have focused on
E-cadherin as a WT1 target gene, due to its close association with
epithelial differentiation.
E-cadherin is a calcium-dependent cell adhesion protein
localized at the adherens junction that mediates cell-cell
interactions. Expression of E-cadherin serves as a marker for
epithelial differentiation (27, 28). The intracellular portion of
E-cadherin interacts with submembrane cytoskeletal proteins including
the - and 
-catenins (27-31). Loss of the E-cadherin expression
is frequently seen in human tumors (28) and has been associated with
loss of epithelial cell morphology and tumor invasion (32-34). The
expression of E-cadherin and other cell adhesion
proteins also play a key role in normal development during the
mesenchymal-epithelial transition (35, 36). E-cadherin appears in the
induced metanephric mesenchyme once condensation into epithelia begins
(37, 38), the same time at which WT1 expression rises in the developing
kidney (39, 40).
In this report, we provide evidence that the WT1 protein can directly
trans-activate E-cadherin gene expression. Both transient and stable
expression of WT1 induced the endogenous E-cadherin gene. WT1 protein
bound to a proximal GC-rich sequence in the E-cadherin gene promoter.
This cis-acting element alone was sufficient for trans-activation of
the E-cadherin promoter by WT1 in co-transfection experiments. A
dominant negative mutant of WT1, WTAR inhibited the E-cadherin promoter
in WT1 expressing NIH 3T3 cells. E-cadherin represents a downstream
target gene that may link the functions of WT1 in epithelial
differentiation and tumor suppression.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
The NIH 3T3 cells stably expressing WT1 (WR16
and WR35) and the Ras-transformed NIH 3T3 cells stably expressing WT1
(VW9) that were used in this study have been described previously (13, 26). Ecotropic PHOENIX cells (gift of W. Pear, University of Pennsylvania, Philadelphia, PA) used to generate retroviral stocks were
grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum.
Plasmid Construction--
Expression vectors for wild-type WT1
isoforms (A, B, C, D) and the WT1 (AR) dominant negative mutant were
described previously (13, 41-43). The promoter regions of the murine
E-cadherin gene were amplified from murine genomic DNA by
PCR2 using various forward
primers containing a BglII adaptor sequence to generate
deletions or point mutations. Mutated bases are indicated in bold type.
A reverse primer containing a HindIII adaptor sequence at
+113 of the murine E-cadherin promoter sequence was used (Sequences 1-17). The human E-cadherin promoter luciferase construct HECad (
368/+125) was described previously (44). For the construction of
HECad (217/+125) and HECad (DEL), E-cadherin promoter sequences were
amplified by PCR and subcloned into SacI and
HindIII of pGL2-Basic (Promega, Madison, WI). A bicistronic
retroviral vector for WT1 was constructed by insertion of a
Sau3A fragment of WT1(A) into the BglII site of
the MIGR1 retroviral vector (gift of W. Pear), which contains the
murine stem cell leukemia virus long terminal repeat.
A single reverse primer (R) containing a BglII adaptor was
paired with each forward primer (F).
The polymerase chain reaction was performed at a denaturing
temperature of 94 °C for 30 s, annealing temperature of
60 °C for 30 s, and extension for 1 min at 72 °C for 40 cycles. After amplification, the PCR fragments were digested with
BglII and HindIII and cloned into the pGL3-Basic
luciferase reporter construct (Promega), and subjected to automated DNA sequencing.
Transfection Assay--
For cotransfection assays involving the
murine and human E-cadherin luciferase reporter and WT1 isoforms (A, B,
C, D), NIH 3T3 cells and 293T cells were grown in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum and
penicillin/streptomycin. At 18 h prior to transfection, NIH 3T3
cells were plated at a density of 3 × 104 in 24-well
dishes. Optimal quantities of effector (RSV-WT1(A, B, C, D), WTAR) and
reporter (E-cadherin-Luc) used in transfection assays were empirically
determined to be 0.5 µg of effector (RSV-WT1(A, B, C, D) or RSV
alone), 0.1 µg of reporter, and 0.01 µg of a thymidine kinase
Renilla reporter gene, which serves as an internal control for transfection efficiency. WT1 modestly activated the thymidine kinase Renilla reporter, leading to an underestimate of
activation by WT1 but did not alter the pattern of activation noted for
E-cadherin promoter deletion constructs. The plasmids were transfected
using the SuperFect transfection reagent (Qiagen, Valencia, CA)
according to the manufacturer's directions. The NIH 3T3 cells were
harvested 60 h after transfection and assayed for luciferase
activity using the Dual-Reporter assay system (Promega). All results
are shown as the average ± standard deviation of three to six
independent experiments. For preparation of nuclear extracts, 293T
cells were plated at the density of 5 × 106
cells/10-cm dish 18 h prior to transfection and 5 µg of
RSV-WT1(A) was used for transfection. The 293T cells were harvested
48 h after transfection, and nuclear extract was prepared as
described below.
Generation of Retrovirus--
A bicistronic vector harboring the
green fluorescent protein and WT1(A) cDNAs was transiently
transfected using Superfect (Qiagen) into PHOENIX cells. At 48 h
after transfection, expression of the vector was monitored by phase
contrast fluorescence microscopy (Nikon, Tokyo, Japan) and the
supernatant of the packaging cells was collected, filtered, and layered
in the presence of 5 µg/ml Polybrene (Sigma) on a fresh culture of
60% confluent NIH 3T3 cells. At 24, 48, and 72 h after infection,
the cells were monitored for green fluorescence and lysed for the
preparation of total RNA using Trizol (Life Technologies, Inc.).
Analysis of mRNA Expression--
Cytoplasmic RNAs were
isolated from cell lines as described previously (13, 26). Cytoplasmic
RNAs from each cell line (20 µg) were subjected to Northern blot
analysis for E-cadherin expression (45) and quantified by
PhosphorImager analysis using ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). The E-cadherin probe was a gift from S. Mazur (Mount
Sinai School of Medicine, New York, NY). E-cadherin expression in
retrovirus-infected NIH3T3 cells was detected by reverse
transcriptase-PCR. The reverse transcriptase reaction was performed
with 1 µg of total RNA using Superscript II, (Life Technologies,
Inc.). The cDNA was then subjected to 35 rounds of PCR using
E-cadherin-specific primers (E-cadherin forward PCR primer, 5'-TCA ACG
ATC CTG ACC AGC AGT TCG-3'; reverse transcriptase primer, 5'-GGT GAA
CCA TCA TCT GTG GCG ATG-3'; reverse nested PCR primer, 5'-ATC TTC TGA
TCC ATG ACC GTG TCC G-3'). Polymerase chain reaction was performed at a
denaturing temperature of 94 °C for 30 s, annealing temperature
of 60 °C for 30 s, and extension for 1 min at 72 °C for 35 cycles. An internal control for RNA integrity was simultaneously
performed using mouse -actin-specific primers (forward, GCC CAG AGC
AAG AGA GGT AT; reverse, GGC CAT CTC TTG CTC GAA GT). The PCR products
were visualized by agarose gel electrophoresis and ethidium bromide staining.
Electrophoretic Mobility Shift Assay (EMSA)--
Double-stranded
oligonucleotide probes were created by annealing complementary
single-stranded oligonucleotides (Life Technologies, Inc.). The
oligonucleotide sequences used were as follows. The putative WT1
binding site is underlined, and mutated position(s) are indicated in
bold type.
The HindIII 5' overhanging ends of the duplex
oligonucleotides were labeled with [-32P]dCTP and the
Klenow fragment of DNA polymerase I and gel-purified. In
vitro coupled transcription/translations were performed with pSP64-WT1(A) (41) or an insertless pSP64 control vector using rabbit
reticulocyte lysate (Promega). Nuclear extract from WT1(A)-expressing cell line VW9 (13), WT1(A)-transfected 293T, and a NIH 3T3 control cell
line were extracted using a two-step method as follows. 5-10 × 106 cells were grown to 80% confluence and resuspended in
10 mM HEPES (pH 7.9), 1.5 mM MgCl2,
10 mM KCl, 0.5 mM DTT. The cells were allowed
to swell on ice for 10 min, vortexed, and briefly pelleted in a
microcentrifuge at 4 °C. The pellet was resuspended in 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT and incubated on ice for 20 min. The nuclear extract
was then centrifuged for 2 min in a microcentrifuge at 4 °C, and the
supernatant was collected. Protein concentrations were determined using
the Bio-Rad DC kit. To compare the binding activity of the alternative
+KTS and KTS forms of WT1 to the E-cadherin promoter WT1-response
element, sequences encoding the zinc-finger domains of WT(A/KTS),
WT(C/+KTS), and WT(AR) (negative binding control) were cloned into the
pGEX2TK GST-expression vector (Amersham Pharmacia Biotech), expressed
in Escherichia coli strain BL21 and purified on
glutathione beads (1).
DNA binding reactions using in vitro transcribed/translated
WT1 were performed with 2.5 µl of lysate programmed with pSP64-WT1(A) or the pSP64 control as described previously (46), except that the
total volume of the reaction was 20 µl and the amount of probe used
was 0.2 ng (approximately 106 cpm/ng). Binding reactions
utilizing nuclear extracts were performed by preincubating 3 µg of
extract in 20 mM HEPES (pH 7.5), 12% glycerol, 1 mg/ml
bovine serum albumin, and 0.5 mM DTT with 2 µg of d(I-C)
and 1 µg of DNase-free RNase A for 15 min at room temperature. Next,
0.2 ng (approximately 106 cpm/ng) of radiolabeled probe was
added and the mixture was incubated for an additional 20 min at room
temperature. Binding reactions utilizing GST-WT1 protein were performed
by preincubating 200 ng of protein in 50 mM HEPES (pH 7.4),
50 mM KCl, 50 µM ZnCl2, 5 mM MgCl2, 1 mM DTT, and 20%
glycerol, with 1 µg of d(I-C) for 10 min at room temperature. After
this, 0.2 ng (approximately 106 cpm/ng) of radiolabeled
probe was added and the mixture was incubated for 30 min at 4 °C.
The DNA-protein complexes were separated through a 5% non-denaturing
polyacrylamide gel for 1.5 h at 300 V at room temperature. For
Western blot analysis following the mobility shift assay, the assay was
performed with an unlabeled oligonucleotide probe. The polyacrylamide
gel was transferred to an Immobilon-P nylon membrane (Millipore,
Bedford, MA), and immunoblotting analysis was performed as described
below. The relative amount of DNA bound by WT1 protein was quantified
utilizing a PhosphorImager and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Immunoblotting--
Cell lines were grown to 80%
confluence in 10-cm plates, scraped into phosphate-buffered saline
(PBS), and centrifuged at 1000 × g. The cell pellets
were resuspended in 200 µl of PBS and solubilized in 200 µl of 2×
Laemmli buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol)
for 1 h at 4 °C, The protein samples were then incubated at
100 °C for 10 min and centrifuged in a microcentrifuge at 4 °C
for 15 min, and the resulting supernatant was collected. Protein
concentrations were determined using the Bio-Rad DC kit. -Mercaptoethanol was added to the samples to a final concentration of 0.5 M, and then heated at 85 °C for 10 min prior to
loading on a gel. Protein samples (40 µg) were separated by
electrophoresis through a 12% SDS-polyacrylamide gel and transferred
to an Immobilon-P (Millipore, Bedford, MA) nylon membrane. The filters
were blocked with 5% dry milk in PBS for 2 h, incubated with a
rabbit polyclonal anti-WT1 antibody (C19, Santa Cruz Inc.) at 200 ng/ml, or with a rat monoclonal anti-mouse E-cadherin antibody (ECCD-2,
Zymed Laboratories Inc., San Francisco, CA) at 1 µg/ml in 0.5% dry milk Tris-buffered saline with 0.05% Tween 20 for
1 h, rinsed in Tris-buffered saline with Tween 20, and probed with
a horseradish peroxidase conjugated anti-rabbit (Roche Molecular
Biochemicals) (1:7500 dilution) or anti-rat (Santa Cruz Biotechnology)
(1:1000 dilution) secondary antibody for 1 h. After rinsing in
Tris-buffered saline with Tween 20, the immunoblots were developed by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
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RESULTS |
Induction of E-cadherin Expression in NIH 3T3 Cells by Stable
Transfection and Retroviral Infection--
In previous studies, we
created a number of normal (WR16 and WR35) and Ras-transformed (VW9)
NIH 3T3 cell lines stably expressing WT1 isoform A (17 amino acids,
KTS). In these cells, a number of epithelial markers were
consistently up-regulated, including the E-cadherin gene (Fig.
1A and Ref. 26). To examine if
the endogenous E-cadherin gene is directly and immediately regulated by
WT1(A), NIH 3T3 cells were infected with a bicistronic retrovirus carrying WT1(A) and GFP (Fig. 1B). E-cadherin mRNA was
induced in NIH 3T3 cells as early as 24 h after infection with a
retrovirus harboring WT1(A) and continued to increase at 48 and 72 h after infection. No induction of E-cadherin was noted in a control
culture infected with a virus containing only GFP. These results
suggested that E-cadherin induction was a direct result of WT1
expression rather than an effect of clonal selection.
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Fig. 1.
E-cadherin expression is up-regulated in
association with stable expression of WT1. A, Western
analysis (top) and Northern analysis (middle) of
E-cadherin expression in NIH 3T3 cells stably expressing WT1(A).
WT1-expressing WR16 and 35 are previously described (26) NIH 3T3 cell
lines stably transfected with RSV-WT1(A). 18 S ribosomal RNA was used
as an internal control for Northern analysis. B, acute
expression of WT1 induces the endogenous E-cadherin gene. NIH 3T3 cells
were infected with a retrovirus harboring green fluorescent protein or
a bicistronic virus harboring GFP and WT1(A). mRNA from the
infected cultures was isolated at 24, 48, and 72 h after infection
and subjected to reverse transcriptase-PCR analysis for E-cadherin
expression. -Actin expression was used as an internal control.
C, minus reverse transcriptase control from
GFP-WT1(A)-infected NIH 3T3 cells.
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Transcription of the Mouse and Human E-cadherin Promoter Is
Activated by WT1--
In order to determine if WT1 directly regulated
the E-cadherin gene, we tested the ability of WT1 to activate both the
mouse and human E-cadherin promoters. Previous studies indicated that the proximal promoter region of murine E-cadherin (178 to +113) is
required for epithelial expression (47-49) (Fig.
2). Within the proximal promoter there
are three regions that contribute to epithelial expression (Fig.
2A) (50, 51): 1) a palindromic element (Pal/Ap2) located at
(98 to 78), which stimulates transcription in epithelial cell and
represses E-cadherin expression in mesenchymal cells; 2) a CAAT box
(65 to 61); and 3) two G-C rich regions I (52 to 44) and II
(41 to 32), which confer basic epithelial promoter activity. A
WT1/EGR-1 binding site (CCGGGGGCG) was found at position 51 to 43
of the murine E-cadherin promoter (Fig. 2A), suggesting that
WT1 could directly regulate E-cadherin. Sixteen different murine
E-cadherin promoter fragments were tested for their response to the WT1
protein (Fig. 2B). Initially, the full-length proximal
(178) reporter construct was co-transfected into NIH 3T3 cells with
vectors expressing WT1 from the Rous sarcoma virus long terminal repeat
(Fig. 3). All four major isotypes of WT1 (A, B, C, D) strongly activated the E-cadherin promoter. Particularly striking activation was noted in response to WT1(A) and WT1(B), the
isoforms of WT1 that lack the KTS sequence between zinc fingers 3 and
4, while isoforms C and D (+KTS) stimulated the promoter to a lesser
extent. Since our previous studies were performed using the WT1(A)
protein, we continued analysis with this isoform.
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Fig. 2.
E-cadherin promoter epithelial-specific
sequence. A, schematic representation of the 5' region
of the murine E-cadherin gene required for epithelial-specific
expression (178 to +113) and comparison of the human and murine
proximal E-cadherin promoter sequences. The E-palidromic/AP-2 binding
site, CAAT box, and GC-I/WT1 site are indicated by gray
shading. The intermediate sequence between the CAAT box and
GC-I, INT, is depicted with a black
bar. All luciferase constructs were created based on a
PCR-amplified fragment of this segment. B, deletions and
point mutants of the murine E-cadherin promoter were created by PCR
amplification of mouse genomic DNA using different forward primers as
described under "Experimental Procedures" and a fixed +113 reverse
primer. The CAAT box and putative WT1 binding consensus are
underlined.
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Fig. 3.
Transcription of the murine E-cadherin
promoter by the different isoforms of WT1. A murine E-cadherin
promoter/luciferase construct (0.1 µg) was transiently co-transfected
into NIH3T3 cells along with an expression vector for each of the four
major WT1 spliced isoforms (WTA, WTB, WTC, WTD) (0.01, 0.1, 0.5, and 1 µg) and a Renilla luciferase internal control vector (0.01 µg). At 60 h after transfection, a dual luciferase assay was
performed. Normalized luciferase expression ± S.D. of triplicate
experiments is plotted. White bar, 0.01 µg; pale
gray bar, 0.1 µg; dark gray bar, 0.5 µg;
black bar, 1 µg.
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Four deletion constructs of the E-cadherin promoter were utilized to
test the importance of the three regions in E-cadherin promoter that
contribute to epithelial expression. The E-cad(178/+113) construct
contains wild-type proximal promoter elements which, as described
previously, are sufficient-specific for epithelial expression (47). The
E-cad(74/+113) and E-cad(67/+113) constructs are deleted for the
palindromic element (E-pal), but retain a CAAT box and a GC-rich
potential WT1 binding site consensus sequence. The E-cad(61/+113)
construct is deleted for the E-Pal and CAAT box, but retains the GC
sequence. The E-cad(61/+113[GCm]) construct harbors a point
mutation in the GC/WT1 element. The (30) construct is a complete
deletion of the three E-cadherin epithelial responsive elements (Fig.
2).
As shown in Fig. 4A, the
full-length construct (178), was activated about 15-fold by WT1(A) in
NIH 3T3 cells relative to a control transfection with the insertless
RSV expression vector. The (74) and (67) constructs, which were
deleted for the E-pal element, were still strongly activated by WT1(A)
(25- and 15-fold, respectively), which showed that E-pal segment is not
important for WT1-mediated activation. Deletion of the CAAT box and GC
elements (30) significantly abrogated the ability of WT1(A) to
activate reporter gene activity. Therefore, the minimal set of
cis-acting sequences responsive to WT1(A) included the GC-I
box (61). Consistent with this view, when the GC-I sequence was
mutated from CCGGGGGCG to CCGGGTGCG, the ability of the
(61) reporter construct to be activated by WT1 was abolished in a
manner similar to deletion of the GC-I sequence (i.e. the
30 construct) (Fig. 4A). It must be noted that the
deletion mutations of the E-cadherin promoter, in addition to
eliminating the ability of WT1 to activate transcription, severely
impaired the basal activity of the promoter. This may be due to
elimination of GC-rich sequences, which can bind the widely expressed
Sp1 and CAAT-binding transcription factors. Potentially WT1 may need to
cooperate with such basal elements and transcription factors in order
to activate gene transcription.
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Fig. 4.
Activation of the murine E-cadherin
promoter by WT1. A, deletion analysis of the murine
E-cadherin promoter identifies a GC-rich WT1-response element. NIH 3T3
cells were transfected with 0.5 µg of a RSV control expression vector
(white bar) or 0.5 µg of RSV-WT1(A)
(black bar) along with 0.1 µg of the indicated
E-cadherin promoter construct. A Renilla luciferase
expression vector (0.01 µg) was used to normalize for transfection
efficiency. The average normalized luciferase activity ± S.D. of
triplicate experiments is presented. B and C,
fine mutational analysis of the proximal E-cadherin promoter
(nucleotides 67/+113) to determine sequences required for full
activation by WT1.
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Constructs 74 and 67 had the strongest response to WT1, which
suggested that both the CAAT box and the GC-I region were essential for
full activation. Therefore, we next performed a fine mutational
analysis of the region between 67 and 44 to pinpoint the
WT1-responsive element(s). For convenience, we divided the region into
three subregions (C is the CAAT box; I is the intermediate region
between CAAT box and GC-I; and GC is the GC-I site) (Fig.
2B). Whereas the wild-type 74 construct was activated about 15-fold by co-expression of WT1, constructs harboring point mutations in either the GC-I element or the CAAT box ((C-I-GCm), (C-I-GCmm), and (Cm-I-GC)) (Fig. 4B) could still be induced
4-8-fold by expression of WT1. However, constructs with mutation of
both the CAAT box and GC-I sequence, (Cm-I-GCm) and (Cm-I-GCmm), no longer yielded appreciable transcription in the presence of WT1 (Fig.
4B).
Our findings did not rule out the possibility that intermediate
sequences, including nucleotides "GCGG" located between the CAAT
box and GC-I site were able to substitute for the loss of the GC-I site
and that the observed response of the CAAT element to WT1 was really
due to a second cryptic WT1-response element. To test this hypothesis,
the GCGG sequence was drastically altered to TTTT and co-transfection
analysis with WT1 was performed (Fig. 4C). When the CAAT box
and GC-I sites were intact, mutation of the intermediate sequence
(C-Im-GC) alone did not affect the ability of the construct to be
induced by WT1. Mutation of the intermediate sequence in combination
with either a mutant CAAT box (Cm-Im-GC) or mutant GC-I site (C-Im-GCm,
C-Im-GCmm) (Fig. 4C) did not drastically affect the reporter
activity (Fig. 4, compare B and C). When the CAAT, intermediate, and GC-I sequences were simultaneously mutated (Cm-Im-GCmm in Fig. 4C), response to WT1 was lost.
Collectively, our findings indicate that WT1 mediates activation of the
murine E-cadherin promoter through the GC-I element and the CAAT-box (see Table I). The GC element was
sufficient to mediate significant induction of the promoter, but the
combination of the two sites led to an additive effect on transcription
in the presence of WT1.
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Table I
Summary of WT1 binding and WT1-mediated activation in various
mutants in putative WT1 binding site of the E-cadherin promoter
region
 , sequence deleted; w, wild-type sequence; m, single nucleotide
mutants; mm, multiple nucleotide mutants; 0, no activation by WT1; +,
less than half-maximal activation by WT1; ++, half activation by WT1;
++++, full activation by WT1.
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The human and murine E-cadherin promoters are very similar (47, 49),
and the GC element and CAAT box are conserved. The human E-cadherin
promoter responded to WT1 in a pattern qualitatively similar to the
murine promoter (Fig. 5). Expression from
a reporter containing sequences from 368 to +125 of human promoter
HE-cad (368/+125) was activated about 3-fold by WT1(A) relative to
RSV control transfection, while in this set of experiments the murine promoter was activated 5-fold.
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Fig. 5.
Activation of the human E-cadherin promoter
by WT1. NIH 3T3 cells were transfected with 0.5 µg of a RSV
control expression vector (white bar) or 0.5 µg
of RSV-WT1(A) (black bar) along with 0.1 µg of
the indicated human E-cadherin promoter construct. The HECad (Del) is
devoid of all three major promoter elements (Pal/Ap2, CAAT, and GC-I)
and is similar to 30 murine E-cadherin promoter construct. A
full-length murine E-cadherin promoter construct (MECad 178) was used
as a positive control. A Renilla luciferase expression
vector (0.01 µg) was used to normalize for transfection efficiencies.
The average normalized luciferase activity ± S.D. of triplicate
experiments is presented.
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HE-cad (217/+125), which still contains the essential elements of the
human E-cadherin promoter, was activated to a similar extent as the
full proximal promoter. HE-cad(DEL), a construct in which all three
critical elements of the promoter (Pal/Ap2, CAAT, and GC-I) were
deleted, was not responsive to WT1. The CAAT box region and GC-I are
well conserved between human and mouse, but the Pal/Ap2 region is not,
which suggests the importance of CAAT and GC-rich region in E-cadherin
gene regulation (Fig. 2A) and the potential evolutionary
conservation of a WT1/E-cadherin regulatory circuit.
Disruption of E-cadherin Expression by a Dominant Negative Form of
WT1--
Dominant negative form of WT1 mutants were previously
identified both in tumor specimens as well as in patients with the
Denys Drash syndrome (2). We showed that one particular mutant, WT1(AR) (43), which is deleted for exon 9 sequences encoding the third zinc
finger motif of WT1, inhibited the ability of wild-type WT1 to activate
transcription in a cotransfection assay (41). Therefore, we determined
if WTAR could disrupt the activation of E-cadherin in cells stably and
transiently expressing wild-type WT1. In a WT1-expressing NIH 3T3 cell
line (WR16), E-cadherin promoter activity was approximately 5 times
higher than in control NIH 3T3 cells that do not express WT1 (Fig.
6A). However, when WR16 cells
were transiently transfected with the E-cadherin promoter along with a
vector expressing the WT1(AR) mutant allele, E-cadherin promoter activity decreased by over 80% to a level similar to that obtained in
control NIH 3T3 cells (Fig. 6B). When WTAR was transiently cotransfected with WT1(A) in the NIH 3T3 cells, WTAR blocked the WT1A-mediated activation of the E-cadherin promoter by more than 50%
(Fig. 6C).
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Fig. 6.
A dominant negative WT1 mutant inhibits the
E-cadherin promoter. A, the E-cadherin promoter is
stimulated in WT1-expressing NIH 3T3 cells. The E-cadherin luciferase
construct (0.1 µg) was transfected into parental NIH 3T3 cells or
WT1-expressing WR16 cells along with 0.01 µg of the
Renilla luciferase control expression vector to normalize
transfection efficiency. B, the E-cadherin promoter (0.1 µg) was transfected into WR16 cells along with an insertless RSV
expression vector or RSV-WT1(AR) (0.5 µg) encoding a dominant
negative form of WT1. C, the E-cadherin promoter (0.1 µg)
was co-transfected into NIH 3T3 cells with RSV-WTA (0.5 µg) in the
presence or absence of RSV-WT1(AR) (0.5 µg). In all experiments,
normalized luciferase activity was determined 60 h after
transfection. The average normalized luciferase activity ± S.D.
of triplicate experiments is presented.
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WT1 Binds to the GC-I Element of the E-cadherin Promoter--
As
shown above, deletion analysis of the murine E-cadherin promoter
indicated that a GC-rich, EGR-1-like element and the CAAT box were
required for WT1-mediated activation. To demonstrate a direct
interaction between WT1 protein and these promoter sequences, a
electrophoretic mobility shift assay (EMSA) was performed. An oligonucleotide corresponding to nucleotides 62 to 24 of the E-cadherin promoter, which contains the GC-I/WT1 binding site, was
incubated with in vitro transcribed and translated WT1 and subjected to electrophoresis. A DNA-protein complex was formed when the
oligonucleotide was incubated with WT1-programmed rabbit reticulocyte
lysate (Fig. 7A,
lane 2, lower arrow), but
not with unprogrammed lysate (lane 1). This
complex was specifically retarded by a WT1 antibody (Fig.
7A, lane 4, upper
arrow) but was unaffected by a control antibody, G4
(lane 6). No WT1/DNA complex was formed on a
mutant oligonucleotide representing the same GCm sequence that disabled
the response of the murine E-cadherin promoter to co-expressed WT1
(data not shown). Unlabeled GC oligonucleotides efficiently competed
for WT1 binding to the wild-type probe, while the GCm sequence could
not effectively compete for binding even when present in the reaction
at a 2000-fold molar excess (Fig. 7B).
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Fig. 7.
Electrophoretic mobility shift and antibody
supershift analysis using in vitro translated WT1(A)
with GC-I/WT1 sequence. A, an oligonucleotide
representing the 62/24 region of E-cadherin promoter, which
contains the putative WT1 binding consensus (lanes
1-6), or a mutant oligonucleotide (lanes
7-12) containing a G to T transversion was incubated with
control reticulocyte lysates (C) or in vitro
translated WT1 (W). Antibodies used for supershift assay
were anti-WT1 (C19) (lanes 3 and
4) and an anti-Gal4 (G4) antibody as a negative
control (lanes 5 and 6). B,
competition EMSA showing specificity of WT1 binding. In
vitro translated WT1 was allowed to bind to the GC element in the
absence of competitor or in the presence of the indicated -fold excess
of wild-type or mutant GC sequence from the E-cadherin promoter.
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Since all four isoforms of WT1 could transactivate the E-cadherin
promoter (Fig. 3), we tested whether both the + and KTS forms
of WT1 could bind to the GC-I/WT1-response element. GST fusion proteins
containing the four WT1(A/KTS) zinc fingers, WT1(C/+KTS) zinc
fingers, and three zinc fingers of the dominant negative/DNA
binding-deficient WT(AR) mutant were allowed to bind an oligonucleotide
containing the GC and CAAT elements of the E-cadherin promoter (Fig.
8). As expected (43), GST-WT1(AR) did not
bind to DNA. GST-WT1(A/KTS) bound relatively strongly to the promoter
sequence, while WT1(C/+KTS) yielded 1/10 the amount of DNA-protein
complex. This is consistent with transfection data, which showed that
WT1(C) and WT1(D) have a reduced but definite capacity to activate the
E-cadherin promoter (Fig. 3).
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Fig. 8.
Electrophoretic mobility shift to compare the
binding of GST-WT1(A/KTS) and GST-WT1(C/+KTS) to WT1-responsive
sequences. An oligonucleotide representing nucleotides 67 to
24 of the E-cadherin promoter, which contains both the CAAT box and
GC-I WT1 binding site, was incubated with GST-WT1(A/KTS) zinc finger,
WT1(C/+KTS) zinc finger, or WT(AR) dominant negative zinc finger. The
anti-WT1 C-19 antibody was used for supershift analysis.
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To map the WT1 binding element within the E-cadherin promoter,
oligonucleotides containing both the CAAT box and the GC-I/WT1 binding
site (Sequences 18-27) as well as mutant versions of these sites were
utilized in an EMSA with in vitro translated WT1(A) (Fig.
9). Since the CAAT box and the GC-I site
recognize many widely expressed transcription factors, it was necessary
to add a WT1 antibody to the reaction to more clearly identify the
WT1/DNA complex (Fig. 9, A-D, lane 1,
top arrow). A WT1 antibody supershift complex was
observed only when oligonucleotides containing the wild-type GC-I/WT1
binding site were used as probes (Fig. 9, A (lanes 5 and 9), and B
(lane 5)). Mutations in the CAAT box or the
intermediate sequence still allowed the WT1 protein to bind to GC-I
site (Fig. 9, A (lane 9) and
B (lane 5)). An oligonucleotide containing the CAAT box alone did not have any WT1 binding activity (Fig. 9B, lanes 7-10).
Oligonucleotides with a wild-type CAAT box and mutant GC-I site (Fig.
9, C and D, lanes 3-6) did not bind
to WT1 even though the CAAT box could mediate transactivation in
response to WT1. To show that WT1 produced in transient transfections could bind the GC element, we attempted mobility shift assays utilizing
extracts from transfected cells but a large number of background
complexes were noted, potentially representing proteins such as SP1 and
Ap2 that can also bind to GC-rich elements. To determine if WT1 was
among these complexes, we performed an EMSA with extract from 293T
cells transfected with RSV-WT1(A). followed by transfer of the gel for
immunoblot analysis (Fig. 10). A
WT1-DNA complex signal was detected only in 293T cell nuclear extracts transfected with WT1 (lanes 3 and 4)
and not in NIH 3T3 cell nuclear extract (Fig. 10, lanes
1 and 2) or mock-transfected 293T cell extract
(Fig. 10, lanes 5 and 6). A summary of
DNA binding activity and transcriptional response of the mutant
promoter constructs is presented in Table I.
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Fig. 9.
Electrophoretic mobility shift and antibody
supershift analysis using in vitro translated WT1(A)
with CAAT-GC-I sequence. Oligonucleotides representing nucleotides
67 to 24 of the E-cadherin promoter, which contains both CAAT box
and GC-I WT1 binding site and its mutant versions, were incubated with
in vitro translated WT1 (W) or unprogrammed
lysate (C). Antibodies used for supershift assay were
anti-WT1 C-19 antibody (C19), while a anti-Gal4 antibody
(G4) was used as a negative control. A, probes
C-I-GC and Cm-Im-GC. B, probes Cm-I-GC and CAAT box alone.
C, probes C-Im-GCmm and Cm-Im-GCmm. D, probes
C-I-GCmm and Cm-I-GCmm.
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Fig. 10.
EMSA/Western analysis of WT1 protein bound
to GC-I/WT1 binding site. An EMSA assay was performed allowing an
unlabeled 62/24 oligonucleotide to interact with nuclear extracts
derived from NIH 3T3 cells (lanes 1 and
2), mock-transfected 293T cells (lanes
5 and 6), and 293T cells transfected with a WT1
expression plasmid (lanes 3 and 4).
The DNA-WT1 complex was separated by non-denaturing gel electrophoresis
and transferred to a nylon membrane, followed by immunoblot with an
anti-WT1 antibody.
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DISCUSSION |
Although it had been shown that WT1 protein could activate or
repress the expression of a number of promoters in co-transfection experiments (2), then endogenous forms of many of these putative target
genes were later shown not to be regulated by WT1 in biological systems
(25, 52). Much of this discrepancy is due to the mode by which WT1
target genes were identified, namely by searching the promoters of
promising candidate target genes for sequences matching the EGR1/WT1
binding site. Given the relatively GC-rich nature of many promoters,
this approach identified a number of putative target gene. True WT1
target genes should have a specific binding site for the protein,
respond to WT1 expression in transient assays, and be activated or
repressed by both transient and stable co-transfection. However, in
addition, the expression of the endogenous gene, not just a promoter
construct should be affected by expression of WT1. Furthermore, a WT1
target gene should be expressed in the developing and adult organism in
a temporal and spatial pattern that is concordant with WT1 expression.
The transcriptional activity of WT1 required for growth control has
been controversial. Recently, we demonstrated that missense mutants of
WT1 defective for transcriptional activation, but competent for
transcriptional repression, failed to suppress cell growth (42). This
indicates that genes whose transcription is activated by WT1 may be
critical for the biological activity of this tumor suppressor.
We have attempted to identify WT1 target genes using a model system in
which engineered expression of WT1 converts the NIH 3T3 fibroblast cell
line into cells with many features of epithelia (26). This system was
used to survey the expression of many epithelial markers without prior
knowledge of the nature of their promoter sequences. E-cadherin was
among those genes up-regulated in WT1-expressing cells. Here we report
that the E-cadherin gene meets criteria for a WT1 target gene
potentially relevant for the roles of WT1 in both inhibiting cell
growth and inducing differentiation. Though up-regulation of E-cadherin
in the original cell lines could possibly be attributed to clonal
selection, we found that E-cadherin mRNA expression was induced
within 24 h of infection of NIH 3T3 cells with a retrovirus
expressing WT1, consistent with a direct effect of WT1.
Deletions and point mutational analysis of the murine and human
E-cadherin promoters and mobility shift assays indicated that an
EGR-1-like cis-acting sequence was necessary and sufficient for WT1-mediated transactivation. We then demonstrated that the GC
element and the CAAT box were required for full and robust WT1-mediated
transactivation of E-cadherin in mesenchymal NIH 3T3 fibroblasts (Fig.
4). Furthermore, a dominant negative mutant of WT1 WT1(AR) inhibited
E-cadherin promoter activity in cells expressing wild-type WT1 (Fig.
6). Finally, we found that WT1 protein derived from nuclear extracts
could bind to the WT1-response element in the E-cadherin promoter (Fig.
10).
It must be noted that detection of WT1 DNA binding activity in nuclear
extracts has been reported in only a few publications (53-55).
Curiously, WT1 also appeared to mediate activation of the E-cadherin
promoter through a CAAT box, because mutation of this element, but not
sequences between the CAAT box and the GC-I element, significantly
impaired the ability of WT1 to activate the E-cadherin promoter.
Furthermore, a construct with a mutation in the GC box, but with an
intact CAAT box, still supported transcriptional activation by WT1
(summarized in Table I). This is consistent with prior information that
WT1 can affect transcription through protein-protein interactions. WT1
was shown to interact with p53 and modulate the transcription of
reporter genes containing only p53 binding sites (56). WT1 also affects
the transcription of the HSP70 promoter, presumably by sequestration of
HSP70 from the heat shock transcription factor, thus leading to the
activation of the heat shock transcription factor (57). The CAAT box
can bind to the CTF/NF1 factors as well as the heteromeric CP1/CP2 factor. Whether WT1 directly binds to these factors to augment their
transcriptional effect or sequesters negative regulators that interact
directly or indirectly with the E-cadherin CAAT box is currently under
investigation. However, the finding that a CAAT box may act as a
WT1-responsive element suggests a novel mechanism of WT1 action and a
broader potential range of target genes.
It was previously reported that epithelial-specific transcription of
E-cadherin required a palindromic sequence as well as two tandem AP-2
transcription factor binding sites within the proximal GC-rich portion
of the promoter, which we have demonstrated can also bind to WT1 (47,
50, 51). Rb and Myc were later shown to activate E-cadherin gene
expression via interaction with AP-2, through the E-Pal and GC-rich
region in epithelial cells, but not in mesenchymal NIH 3T3 cells (58).
Consistent with these data, we found that transfection of Rb did not
activate the E-cadherin promoter and co-transfection of WT1 and Rb did
not yield synergistic or additive activation on E-cadherin expression
in NIH 3T3 cells (data not shown). However, unlike Rb or Myc, WT1 could
transactivate the E-cadherin gene in non-epithelial NIH 3T3 cells. This
suggests an independent pathway for epithelial differentiation exerted by WT1 and is consistent with the notion that WT1 might up-regulate E-cadherin expression in mesenchymal cells during the
mesenchymal-epithelial transition. However, our studies have not
excluded the possibility of inteaction between WT1 and AP-2 or other
proteins bound to the E-Pal site. WT1-mediated activation was most
efficient when the E-Pal element was deleted (Figs. 3 and 4). This
finding suggests the presence of a negative regulator bound to the
E-Pal element in mesenchymal cells, consistent with previous findings
(47, 50, 51). Such a protein might act to counter WT1-mediated activation of E-cadherin in certain tissues.
E-cadherin expression is characteristic of epithelia and is critical
for epithelial development (27). E-cadherin is required for
embryogenesis, as targeted disruption of this gene leads to failure of
early murine development due to a defect in embryo compaction (59, 60).
More specifically, in the kidney, E-cadherin expression is induced in
the condensing metanephric mesenchyme during the mesenchymal-epithelial
transition (37, 38, 61) at the same time that WT1 is up-regulated in
this tissue (39, 40). This fact, along with the other evidence
outlined, makes a compelling case for E-cadherin as a direct WT1 target
gene. Direct transactivation of E-cadherin by WT1 demonstrates an
evident link between the tumor suppressive activity of WT1 and a gene that plays a major role in differentiation. It confirms our previous report that WT1 plays a direct role in both the induction of the epithelial phenotype and inhibition of tumorigenesis by controlling the
expression of a subset of genes encoding molecules that control epithelial characteristics (26). E-cadherin might mediate many facets
of growth suppression by WT1. Homotypic interactions mediated by
E-cadherin lead to increased cell adhesion and decreased invasiveness. Furthermore, E-cadherin was recently demonstrated to have an
anti-proliferative effect of its own, correlated with the up-regulation
of the CDK inhibitor, p27 (62). We checked for p27 up-regulation after infection of 3T3 cells with the WT1 retrovirus and found that p27 was
expressed at a basal level in 3T3 but was not up-regulated by WT(A)
(Data not shown). This may be due to the relatively low level of
E-cadherin induced after transient expression and does not preclude the
possibility that E-cadherin expression in the developing kidney may
affect the expression of p27 or other cyclin-dependent kinase inhibitors.
The E-cadherin gene is frequently inactivated in human cancer, and its
down-regulation is particularly associated with the acquisition of an
invasive tumor phenotype (28, 34, 63). Allelic loss of heterozygosity
of E-cadherin has been reported in cervical, prostrate, breast,
hepatocellular, and colorectal carcinomas as well as in Wilms' tumor
itself (28, 64-68). Point mutations of the E-cadherin gene were found
in gastric, renal, lung, colorectal, and breast cancers (28, 66,
69-74). Defects in trans-acting pathways regulating E-cadherin
expression have been suggested in breast cancer (44), and epigenetic
inactivation of E-cadherin gene was also implicated by changes in
chromatin structure and the methylation status of CpG sites within of
the E-cadherin promoter (50). Recently, E-cadherin expression within Wilms' tumors was found only in a limited set of cells resembling renal tubules (75). These tumors co-expressed cytokeratin and vimentin,
underscoring the aberrant differentiation of the tumor. Altered
E-cadherin expression as a result of lost or mutated WT1 could play a
role in the pathogenesis of Wilms' tumors. Furthermore, E-cadherin
complexes with -catenin, a protein involved in transduction of wnt
signaling to the nuclear TCF/LEF transcription factors (76). Activating
mutations of -catenin were recently identified in Wilms' tumor
lending more evidence to the potential importance of disruption of the
E-cadherin/catenin axis in this tumor (77).
In summary, E-cadherin is a gene coordinately regulated with WT1 during
kidney development, whose expression can be activated in response to
stable or transient expression of WT1 and whose promoter can be bound
and activated by WT1. Hence, E-cadherin is a likely a bona
fide target gene of WT1.
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ACKNOWLEDGEMENTS |
We thank Pat Wilson, Chris Burrow, George
Atweh, Melanie McConnell, and Deborah Morrison for critical
discussion of the manuscript.
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FOOTNOTES |
*
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.
§
Supported by the Association Pour La Recherche sur le Cancer.
Scholar of the Leukemia and Lymphoma Society. Supported by
National Institutes of Health Grant CA 59998.
**
To whom correspondence should be addressed: Derald H. Ru
TOP
ABSTRACT
INTRODUCTION
