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*
Johannes-Müller-Institut
für Physiologie,
Humboldt-Universität,
Charité, Berlin, Germany
Klinik für Innere Medizin I,
Humboldt-Universität,
Charité, Berlin, Germany
Beth Israel Deaconess Medical Center, Renal Division, Boston,
Massachusetts.
Correspondence to Dr. Holger Scholz, Johannes-Müller-Institut für Physiologie, Humboldt-Universität, Charité, Tucholskystrasse 2, 10117 Berlin, Germany. Phone: +49-30-2802-6610; Fax: +49-30-2802-6662; E-mail: holger.scholz{at}charite.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WT1 is related structurally and functionally to the early growth response family of zinc finger transcription factors and originally was characterized as a transcriptional repressor (reviewed in reference 4). More recent studies indicate that WT1 also can stimulate gene transcription (5,6,7) and possibly in involved in RNA processing (8). Putative WT1 downstream targets include growth- and differentiation-promoting genes, such as insulin-like growth factor 2 (9), Pax2 (10), amphiregulin (11), and E-cadherin (12), among others (reviewed in reference 13). However, the endogenous transcript levels of most candidate genes with WT1-sensitive promoters failed to correlate with WT1 expression (14), suggesting that not all of the putative targets are normally controlled by WT1.
Expression of the WT1 gene is developmentally regulated in embryonic kidneys. WT1 mRNA and protein levels in the metanephric mesenchyme increase upon induction by the ureteric bud (15). Levels remain high in the renal vesicles and the comma- and S-shaped bodies, where WT1 expression is restricted to the podocyte layer of the differentiating glomeruli (15). Little is known about the physiologic function of WT1 during development. WT1 induced apoptosis in osteosarcoma cells by suppressing the synthesis of epidermal growth factor receptor (16). Because programmed cell death occurs at a large scale in embryonic kidneys (17) and because epidermal growth factor rescued the cultured renal mesenchyme from apoptosis (18), WT1 has been proposed to act as a proapoptotic signal in the developing kidney as well (16). However, direct experimental evidence that WT1 can indeed mediate programmed death of renal embryonic cells is still missing.
To characterize further the function of the WT1 transcription factor, we aimed to identify novel WT1 downstream target genes. Using the approach of educated guessing, we tested the hypothesis that vitamin D receptor (VDR) expression is regulated by WT1. VDR belongs to the steroid superfamily of nuclear receptors and mediates the genomic actions of the active metabolite 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Upon binding to its ligand, VDR heterodimerizes with the retinoid X receptor (RXR), and this complex physically interacts with specific DNA recognition sites that are present in the regulatory regions of putative downstream target genes (reviewed in reference 19).
The following lines of evidence suggested to us that VDR might be a potential target for WT1. First, the promoters of the VDR genes from human and mouse contain several predicted WT1 consensus-binding sites (20,21). Second, a number of studies have shown that 1,25-(OH)2D3 can act as a signal for cell differentiation and also may induce apoptosis (reviewed in reference 22). Finally, VDR mRNA and protein have been detected in embryonic kidneys, thus pointing to a role for the vitamin D system in renal development (23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Luciferase and ß-Galactosidase Assays
The cells were lysed 48 h after the transfection, and luciferase activities
were measured in a luminometer (Lumat LB 9501, Bertholdt, Germany) with the
use of beetle luciferin as a substrate (Promega, Mannheim, Germany).
ß-galactosidase activities were determined spectrophotometrically
(Beckman DU 540 spectrophotometer) with the use of a commercial kit according
to the manufacturer's instructions (Promega). Results shown are averages of
five transfection experiments, each performed in duplicate. P <
0.05 was considered significant (ANOVA).
Determination of Cell Proliferation
Cell proliferation was estimated by counting aliquots of the trypsinized
cells in a Neubauer chamber and by measuring 5-bromo-2'-deoxy-uridine
incorporation into genomic DNA followed by an enzyme-linked immunosorbent
assay detection (Roche Diagnostics, Mannheim, Germany). For this purpose, HEK
293 cells stably transfected either with a wt1 expression construct
or with the empty pCB6+ vector were seeded into 96-well plates at a
density of 104 cells per cm2 with variable
concentrations of 1,25-(OH)2D3 in the medium. The
cultures were incubated for 4 d, and the medium containing
1,25-(OH)2D3 was renewed daily. Data presented are means
± SEM of 5 experiments performed as duplicates.
Measurement of 1,25-(OH)2D3 Concentrations
Concentrations of 1,25-(OH)2D3 were measured in
tissue culture supernatants at different time points (4, 8, 12, 16, and 24 h)
after incubation. The supernatants were delipidated by treatment with a
dextran sulfate/magnesium chloride reagent, followed by immunoextraction with
a monoclonal anti-1,25-(OH)2D3 antibody
(Immunodiagnostics Ltd., Hamburg, Germany). 1,25-(OH)2D3
concentrations in the extracts were measured by RIA as described by the
manufacturer (Immunodiagnostics Ltd.).
RNA Preparation and Northern Blot Hybridization
Isolation of total RNA and Northern blot hybridization were performed as
described elsewhere (9). Equal
amounts of total RNA were pooled from stable clones grown separately.
Sodium Dodecyl Sulfate—Polyacrylamide Gel Electrophoresis
Total cell lysates from subconfluent cultures were prepared by heating the
samples in TBS/1% sodium dodecyl sulfate (SDS) buffer to 95°C for 3 min.
Twenty µg of protein were loaded per lane and transferred after separation
on a 10% SDS—polyacrylamide gel electrophoresis onto
polyvinylidenedifluoride membranes (Amersham Pharmacia Biotech, Freiburg,
Germany) with the use of a semidry blotting apparatus (BioRad,
München, Germany). A polyclonal anti-WT1
antibody from rabbit (WT 180; Santa Cruz Biotechnology, Heidelberg, Germany)
was used at a 1:1000 dilution for immunoblotting. After incubation with a goat
anti-rabbit secondary antibody (1:5000), the reaction products were detected
by enhanced chemoluminescence system (Amersham Pharmacia Biotech). A rat
monoclonal anti-VDR antibody (1:500 dilution; Affinity Bioreagents, Hamburg,
Germany) and a biotinylated goat anti-rat IgG secondary antibody (1:1000
dilution; Dianova, Hamburg, Germany) were used for immunodetection of VDR
protein with the biotin-streptavidin technique.
WT1 Immunostaining of Cultured Mouse Renal Embryonic Cells
After fixation with 3% paraformaldehyde in phosphate-buffered saline, the
cells were permeabilized with 0.1% Triton X-100 and endogenous peroxidase
activities were blocked for 5 min in a solution of 3%
H2O2 in methanol (1:4). After washing in TBS, the cells
were incubated for 16 h at 4°C with a rabbit polyclonal anti-WT1 antibody
(Santa Cruz Biotechnology) diluted 1:150 in TBS with 5% normal goat serum.
This incubation was followed by a 2-h treatment with biotinylated secondary
antibody (goat anti-rabbit, 1:100 in TBS with 1% bovine serum albumin; Vector
Laboratories Inc.) and the streptavidin-peroxidase complex (Sigma,
Deisenhofen, Germany). WT1-positive cells were identified by their brown color
after visualization with diaminobenzidine and hydrogen peroxide (Sigma).
Construction of Reporter Plasmids
A 1451-bp fragment from the mouse vdr promoter (from 70 to 1521 bp
of the published sequence
(20)) was cloned by PCR with
the use of genomic DNA from mouse liver as a template and the following
primers: 5'-TGCCCTAAGGTGTTGGCT-3' (forward primer),
5'-TGGACACACAGCTCGGCG-3' (reverse primer). The PCR product was
ligated into the SmaI/BglII restriction sites of the
pGL2basic reporter plasmid and confirmed by dideoxy sequencing of both
strands. This construct was designated pVDR1451. 5'-deletion
mutants were generated from pVDR1451 by PCR with the use of the
following forward primers: 5'-CGGATCATCACAGGCAGA-3',
5'-TTGAGGGGCAGGGCGGTC-3', 5'-CCAGGTGCTGAGCAGTCT-3'.
The PCR products were cloned into the KpnI/BglII sites of
pGL2basic and designated pVDR306, pVDR105, and
pVDR60, respectively.
Electrophoretic Mobility Shift Assays
Mouse recombinant WT1 protein was generated in the presence of
[35S]methionine with the use of the TNT quick coupled
transcription-translation system (Promega). An aliquot of the translation
product was analyzed on a 10% SDS-polyacrylamide gel, which showed a single
band of the size of WT1 protein (approximately 52 kD). DNA binding reactions
were performed for 30 min at room temperature with 20 ng of the unlabeled
recombinant WT1 protein (+ and - KTS isoforms) in 10 µl of a 1 x
reaction buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM MgCl2,
0.5 mM ethylenediaminetetraacetate, 0.5 mM DTT, 14% glycerol, 0.05 mg/ml poly
[dI-dC]). For the supershift experiments, 1 µg of a polyclonal rabbit
anti-WT1 antibody (C19; Santa Cruz Biotechnology) was added to the reaction
mixture. The end-labeled 21-bp double-stranded oligonucleotide
(5'-TGAACTTAGTGGGCGTGGTTG-3') contained the predicted WT1
element from the proximal mouse vdr promoter. A 28-bp DNA fragment including
the WT1 consensus binding site from the platelet-derived growth factor A-chain
promoter served as a specific competitor
(5'-GGGGCGGGGGCGGGGGCGGGGGAGGGG-3')
(26).
Induction and Analysis of Apoptosis in Cultured Mouse Renal Embryonic
Cells
Primary cultures of kidney cortical cells were prepared from mouse embryos
on gestational day 12 (E12) by an enzymatic digestion procedure as described
previously (27) and grown for
1 d in DMEM-10% FCS. The viable cells were split into 96-well plates at a
density of 103 cells/cm2 and cultured for 72 h in
DMEM-10% FCS supplemented with variable concentrations (1 nM to 1 µM
nominally) of 1,25-(OH)2D3. The active vitamin D
compound (Leo Pharmaceutical, Ballerup, Denmark), which was obtained as a 4
x 10-3 M stock solution, was diluted to 10-6 M in
ethanol. The final ethanol content in the tissue culture medium was 
0.1
vol %. Control experiments were performed by incubation of the cells for 72 h
with the appropriate amounts of ethanol (in DMEM-10% FCS). The following
techniques were applied to identify apoptotic cells: (1) the
characteristic signs of apoptosis, including membrane blebbing, cytoplasmic
shrinkage, and nuclear condensation, were observed at 400x magnification
after hematoxylin and eosin staining of the cultures (Axioplan 2; Zeiss,
Göttingen, Germany); (2) free
3'-OH termini in apoptotic cells were fluorescence-labeled with the
terminal deoxynucleotidyl transferase (TdT) technique with the use of a
commercial kit according to the manufacturer's instructions (Roche
Diagnostics); (3) apoptotic cells were stained with a FITC-conjugated
anti-annexin V antibody (Roche Diagnostics) and visualized under a
fluorescence microscope (400x magnification, Axioplan, Zeiss). The
annexin V immunopositive cells were counted in 10 optical fields from two
dishes each. Only those annexin V—positive cells that did not stain with
propidium iodide were considered apoptotic. The fraction of cells that
exhibited annexin V immunoreactivity was calculated in five independent
experiments and taken as an estimate for the proapoptotic action of
1,25-(OH)2D3.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VDR Expression in Transfected HEK 293 Cells
We performed Northern blot hybridization using a full-length human cDNA
probe to compare VDR mRNA levels in wt1- and
pCB6+-transfected clones. As shown in
Figure 1B, VDR mRNA
content was significantly higher (fivefold by densitometry) in
wt1-expressing than in pCB6+-transfected cells. The
increase of VDR mRNA in wt1-HEK 293 was associated with
upregulation of VDR protein as indicated by immunoblot analysis with a rat
monoclonal anti-VDR antibody (Figure
1D). ß-Actin transcript levels were approximately the same in
the wt1- and pCB6+-transfected clones
(Figure 1C). Because HEK 293
cells that were transfected stably either with wt1 cDNA (in
pCB6+ plasmid) or with pCB6+ empty vector shared a
similar genetic background, the observed differences in VDR gene
expression likely were due to the different WT1 levels.
Activation of the VDR Gene Promoter by WT1
To test whether transcription from the vdr promoter could be
activated by wt1, we transiently co-transfected HEK 293 cells with
wt1 expression vectors (four different splicing variants) and a
construct containing approximately 1.5 kb of mouse vdr promoter
sequence (20) upstream of the
firefly luciferase reporter gene (pVDR1451). Transient
co-transfections were performed because the permanent lines were extremely
difficult to transfect, yielding transfection efficiencies of less than 5%.
Normalized luciferase activities that were taken as a measure for the
transcriptional activity of the vdr promoter are indicated in
Figure 2. Co-transfection of
pVDR1451 (2 µg) along with the different wt1 expression
constructs (15 µg each) stimulated vdr promoter activity more than
fourfold. Luciferase activity of pVDR1451 increased in proportion
with the amount (2 to 15 µg) of co-transfected wt1 expression
plasmid (data not shown). In contrast, transcription from the vdr
promoter was not changed significantly by co-transfection of a
pCB6+ construct that contained the full-length wt1 cDNA in
antisense (WT1 rev.) orientation (Figure
2A). Serial 5'-deletion analysis detected a 201-bp fragment
in the proximal vdr promoter that was required for transcriptional
activation by wt1 (Figure 2B).
This sequence contained a predicted WT1 consensus site (-GNGGGNGNG-) 115 bp
upstream of exon 1 (20).
Electrophoretic mobility shift assays were performed to examine whether
recombinant wt1 protein (+ and - KTS isoforms) could physically interact with
the putative WT1 element. Using the 21-bp oligonucleotide from the mouse
vdr promoter as a probe
(5'-TGAACTTAGTGGGCGTGGTTG-3'), we obtained a single
retardation band with both wt1 isoforms
(Figure 3). A similar
retardation signal was seen with nuclear extracts prepared from the permanent
wt1-expressing clones (data not shown). The shifted band could be
competed with excess amounts of a 28-bp unlabeled oligonucleotide including a
WT1 binding site from the platelet-derived growth factor A-chain promoter
(26). The retardation band was
supershifted upon incubation of the reaction mixture with a polyclonal rabbit
anti-WT1 antibody (Figure 3).
No band shift was obtained with an oligonucleotide containing an Sp1 instead
of a WT1 consensus binding site (data not shown).
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1,25-(OH)2D3 Inhibits the Proliferation of HEK
293 Cells
To test whether renal cell growth might be controlled by a vitamin
D—dependent signaling mechanism, we studied the effect of
1,25-(OH)2D3 on the proliferation of pCB6+-
and wt1-transfected HEK 293 cells. As shown in
Figure 4A, the basal
proliferation rates were significantly lower in the wt1-expressing
lines as compared with the pCB6+-transfected cells. Incubation with
the active metabolite 1,25-(OH)2D3 dose-dependently
inhibited 5-bromo-2'-deoxy-uridine incorporation into chromosomal DNA. A
comparison between the slopes of the growth curves in
Figure 4B reveals that the
wt1-expressing HEK 293 cells were by far more sensitive to the
antiproliferative action of 1,25-(OH)2D3 than the
pCB6+ transfectants. Notably, the
1,25-(OH)2D3 concentrations measured in the tissue
culture supernatants after 24 h of incubation were approximately 15-fold lower
than those nominally adjusted in the medium. This finding is consistent with
an estimated in vitro half-life of 1,25-(OH)2D3
of approximately 6 h (30).
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1,25-(OH)2D3 Induces Apoptosis of
Wt1-Expressing Renal Embryonic Cells
Because the active vitamin D metabolite can induce apoptosis in a variety
of cells (reviewed in reference
22), we examined whether
programmed cell death also was involved in the action of
1,25-(OH)2D3 on embryonic renal cells. As a more
physiologic model than the permanent HEK 293 cell lines, we used renal cells
that were freshly isolated from mouse embryonic kidney cortex. These primary
cultures consisted of more than 80% wt1-immunopositive cells, a significant
fraction of which exhibited the signs of apoptosis (e.g., membrane
blebbing, cytoplasmic shrinkage, chromatin condensation) after a 72-h
incubation with 1 nM 1,25-(OH)2D3
(Figure 5D versus
Figure 5C). Programmed cell
death was confirmed by the demonstration of nuclear DNA fragmentation with the
use of in situ DNA nick end labeling assay
(Figure 5F versus
Figure 5E) and by annexin V
immunocytochemistry. The effect of the active vitamin D metabolite was
dose-dependent, and the fraction of annexin V—positive cells was 45
± 6%, 62 ± 5%, 73 ± 6%, and 82 ± 4% in the
presence of 1 nM, 10 nM, 100 nM, and 1 µM
1,25-(OH)2D3, respectively. Of note, the active vitamin
D metabolite caused apoptosis preferentially of the wt1-expressing
renal embryonic cells, and—by consequence—the fraction of
wt1-immunonegative cells increased from less than 20% to more than 90% after a
72-h exposure to 1,25-(OH)2D3
(Figure 5B versus
Figure 5A).
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VDR and WT1 Expression in the Developing Kidney
Time courses of wt1 and vdr expression were studied by
Northern blot analysis in the developing rat kidney in vivo.
Intrarenal wt1 and vdr transcript levels were closely
related between days 15 and 21 of embryonic development
(Figure 6). Whereas
vdr mRNA levels remained high throughout adulthood, wt1
transcripts were barely detectable in kidneys from adult rats
Figure 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have shown that the active metabolite 1,25-(OH)2D3 acting through the intracellular VDR can promote cell differentiation in addition to its effects on calcium and phosphate metabolism (reviewed in references 19 and 22). Interestingly, some of the molecular targets of VDR, e.g., epidermal growth factor receptor (31) and cyclin-dependent kinase inhibitor p21 (32), also are regulated by the WT1 transcription factor (6,16), indicating that WT1 and VDR act through common intracellular signal transduction pathways. Functional synergism between WT1 and VDR is supported by our observation that WT1 stimulated VDR expression in human embryonic kidney cells. Upregulation of VDR by WT1 most likely occurred at the transcriptional level, because co-transfection of WT1 expression constructs stimulated vdr promoter activity probably through direct interaction of WT1 protein with a predicted consensus sequence in the proximal vdr promoter.
The transcriptional effect of WT1 may depend on the type of expression vector used for transfection (33). Because we transfected cytomegalovirus promoter—based constructs that have been found to repress rather than activate gene transcription (33), it might even underestimate the stimulatory potency of wt1 in our experimental setting. For comparison, a threefold induction of the human forkhead gene promoter (34) and a fivefold activation of the syndecan-1 promoter (5) by WT1 have been reported. The exact mechanism for the dual regulatory functions of WT1 that can either repress or enhance gene transcription is still unclear but may involve specific protein—protein interactions (35,36).
The metanephric blastema and the podocyte precursors of the immature glomeruli are major sites of wt1 expression in the developing kidney (15). Vdr immunopositive cells also have been detected recently in the renal mesenchyme and the visceral and parietal glomerular epithelium of embryonic rat kidney (23). Our findings demonstrate that the temporal expression patterns of wt1 and vdr correlate closely during renal development. It seems likely, therefore, that VDR is a physiologic target gene for WT1 not only in vitro but also in the developing kidney in vivo.
The functional significance of our findings is supported by the observation that the growth inhibitory effect of 1,25-(OH)2D3 was enhanced dramatically in wt1-expressing HEK 293 cells. Accordingly, less than nanomolar concentrations of the active vitamin D metabolite 1,25-(OH)2D3 induced programmed death predominantly of the wt1-immunopositive cells from mouse embryonic kidney cortex. These observations suggest that the sensitivity to the proapoptotic action of 1,25-(OH)2D3 was enhanced in the wt1-expressing cells. Apoptosis seems to be particularly important in renal development as the kidneys were the most severely affected organ in Bcl2 knockout mice (37), and approximately 3% of all cells within the nephrogenic renal cortex are apoptotic at any given time during development (17). Our results suggest that the vitamin D endocrine system contributes to the high rate of apoptosis in the developing kidney. Because 1,25-(OH)2D3 may induce programmed cell death through a VDR-dependent signaling mechanism (38), upregulation of VDR by the WT1 transcription factor therefore would increase the susceptibility of renal embryonic cells to the proapoptotic action of vitamin D metabolite. Similar results have found recently with breast cancer cells, which exhibited increased rates of apoptosis in response to chemotherapeutic agents after pretreatment with 1,25-(OH)2D3 (39).
A role of the vitamin D endocrine system in renal development is seemingly in conflict with the normal embryonic development of vdr-/- mice (40,41). Surprisingly, the vdr null mutant animals were phenotypically normal at birth and did not exhibit the signs of vitamin D deficiency until weaning. After weaning, however, vitamin D—dependent rickets type II and growth retardation developed in homozygous mice, leading to death within 15 wk after birth (40,41). The normal phenotype of the vdr null mice does not exclude a role of the vitamin D system in renal cell differentiation but rather suggests a redundancy in vitamin D—dependent signaling throughout embryonic and early postnatal development. Similar findings were made recently with amphiregulin, a member of the epidermal growth factor family, that has been identified as a transcriptional target of WT1 (11). Thus, amphiregulin stimulated the branching morphogenesis of metanephric kidney explants (11), but no gross developmental defects were observed in kidneys from amphiregulin knockout mice (42).
Taken together, our findings demonstrate that the WT1 gene product transcriptionally activates VDR expression in human embryonic kidney cells. Upregulation of VDR by the WT1 transcription factor may mediate apoptosis of renal embryonic cells in response to 1,25-(OH)2D3. These findings suggest a role for the vitamin D endocrine system in the regulation of renal cell growth and differentiation during development.
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Acknowledgments |
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,25(OH)2D3 was kindly
provided by Dr. L. Binderup (Leo Pharmaceutical Products). This study was
financially supported by a grant from the Deutsche Forschungsgemeinschaft
(Scho 634/3-1). |
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3: Receptor ontogenesis in fetal
renal development. Am J Physiol38
: F419-F428,1995
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) or with the wt1 cDNA
(+KTS isoform) in pCB6+ (
). The relative lengths of the
vdr promoter constructs and the locations of the putative WT1
consensus binding sites are indicated in the schematic drawing. Normalized
luciferase activities are expressed as relative light units (RLU). Values
shown are averages ± SEM of five experiments performed in duplicate.
*, statistical significance (P < 0.05) between
wt1- and pCB6+-transfected cells. Note that deletion of a
201-bp sequence (pVDR306 versus pVDR105)
abolished the wt1 responsiveness of the vdr promoter.
