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Oxygen-regulated expression of the Wilms’ tumor suppressor Wt1 involves hypoxia-inducible factor-1 (HIF-1)¹

KAY-DIETRICH WAGNER^*,2, NICOLE WAGNER^#,2, SVEN WELLMANN, GUNNAR SCHLEY^*, ANJA BONDKE^*, HEINZ THERES^# and HOLGER SCHOLZ^*,3

^* Johannes-Müller-Institut für Physiologie,
Klinik für Pädiatrie mit Schwerpunkt Onkologie und Hämatologie; and
^# Klinik für Innere Medizin I, Humboldt-Universität, Charité,10117 Berlin, Germany

³Correspondence:Johannes-Müller-Institut für Physiologie, Humboldt-Universität, Charité, Tucholskystrasse 2, 10117 Berlin. Germany. E-mail: holger.scholz{at}charite.de

SPECIFIC AIMS

We recently reported de novo expression of the Wilms’ tumor suppressor Wt1, a critical molecule for development of the heart and genitourinary system, in blood vessels of the ischemic myocardium. The purpose of this study was to analyze the mechanism(s) of hypoxic/ischemic induction of Wt1 and to assess the potential impact of oxygen-regulated Wt1 expression.

PRINCIPAL FINDINGS

1. Wt1 mRNA in normoxic and hypoxic organs
RNase protection assay was used to measure Wt1 mRNA in various organs of rats exposed at normal atmosphere (20% O₂) or at normobaric hypoxia (8% O₂). Hypoxic exposure increased Wt1 transcripts in the kidneys and heart on average 3.3- and 2.3-fold (n=5, each) compared with rats at normoxia (20% O₂). A significant increase of Wt1 mRNA was detectable after 2 h of oxygen restriction and could be mimicked by exposure at 0.1% carbon monoxide. No differences in Wt1 mRNA were found in the brain and spleen of normoxic and hypoxic rats.

2. Wt1 is expressed de novo in the tubules of hypoxic kidneys
Immunofluorescent labeling was used to identify sites of Wt1 expression in hypoxic kidneys. Consistent with reports by others, Wt1 was restricted to the nuclei of the glomerular podocytes in normal rat kidney. However, exposure of rats to 8% O₂ for 8 h resulted in an intense cytoplasmic staining of tubules in the renal cortex, which was associated with up-regulation of the hypoxia-inducible factor-1 (HIF-1).

3. Hypoxia is associated with expression of Bcl-2 in renal tubules and a decrease in TUNEL-positive cells
Wt1 has been implicated in the control of cell proliferation and apoptosis. To clarify whether activation of Wt1 in hypoxic rat kidney was related to either function, we performed immunostaining for proliferating cell nuclear antigen (PCNA) and labeling of apoptotic cells with the TUNEL technique. Though no differences in PCNA fluorescence were seen between the kidneys from normoxic and hypoxic rats, on average fourfold fewer TUNEL-positive cells were contained in the renal cortex of rats at 8% O₂ than in normoxic animals (Fig. 1 A, n=3 each). Caspase-3/7 activity that was measured as a second parameter of apoptosis was significantly decreased in hypoxic vs. normoxic kidneys (Fig. 1C ). The decrease of apoptotic cells in hypoxic kidneys was associated with tubular expression of the anti-apoptotic protein Bcl-2, which is transcriptionally activated by Wt1 (Fig. 1B ). Bcl-2 was barely detectable in normoxic kidneys (Fig. 1B ). For comparison, protein levels of p53, an inducer of apoptosis that can be stabilized under hypoxia, were not significantly different in the kidneys of hypoxic vs. normoxic rats (Fig. 1D ).

View larger version (86K): [in this window] [in a new window]   Figure 1. TUNEL labeling of apoptotic cells (A) and immunofluorescent staining of Bcl-2 (B) in normoxic (20% O2) and hypoxic (8% O2) rat kidneys. The number of TUNEL-positive cells was markedly reduced in the renal tissue of hypoxic vs. normoxic rats (A, a,b). Immunofluorescent staining was enhanced in the renal tubules at 8% O2 but virtually absent from normoxic kidneys (B, a, b). No signal was detected upon incubation of the tissue sections with normal rabbit serum instead of anti-Bcl-2 antibody (data not shown). C) Caspase-3/7 activity as a measure of apoptotic cell death in the lysates of normoxic (20% O2) and hypoxic (8% O2) rat kidneys. Values are expressed as relative light units (RLU). *P < 0.05 was considered statistically significant (Student’s t test). Data shown are means ± SE of 4 different kidneys at 20% and 8% O2 each analyzed in duplicate. D) Immunoblot detection of p53 and ß-actin in the kidneys of rats (n=4 each) under normoxia (20% O2) and hypoxia (8% O2).

4. Hypoxia stimulates Wt1 expression in lymphoblasts and osteosarcoma cells
Next we searched for a suitable cell line to study the mechanisms of oxygen-regulated Wt1 expression at the molecular level. Reh lymphoblasts (ATCC CRL-8286) and U-2OS osteosarcoma cells (ATCC HTB-96) responded with an increase in Wt1 mRNA and protein to a 16 h incubation at 1% O₂. Hypoxic induction of Wt1 in these cell lines could be mimicked by treatment with cobalt chloride (CoCl₂) and desferrioxamine (DFX, 100 µM each) at 20% O₂ (Fig. 2 A). CoCl₂ and DFX are both thought to activate hypoxia-inducible genes at normal oxygen tension through deprivation of Fe²⁺, a critical cofactor for HIF-1 prolyl hydroxylase, the rate-limiting enzyme in oxygen-dependent degradation of HIF-1. Activation of Wt1 by hypoxia, CoCl₂, and DFX was associated with an increase of HIF-1 immunoreactivity in these lines whereas ß-actin content remained unaffected (Fig. 2A ).

View larger version (39K): [in this window] [in a new window]   Figure 2. A) Immunoblot detection of Wt1, HIF-1 and ß-actin in U-2OS osteosarcoma cells. Wt1 and HIF-1 content was significantly increased in U-2OS cells after 16 h of incubation at 1% O2. Similarly, treatment with CoCl2 and desferrioxamine (DFX, 100 µM each) at 20% O2 enhanced Wt1 and HIF-1 expression (A). C, control without CoCl2 and DFX, respectively. C, D) The promoter of the mouse Wt1 gene (pWt1prom.) mediates hypoxic induction of a luciferase reporter. U-2OS cells were transiently transfected with the illustrated Wt1 promoter-reporter constructs, which contained two potential HIF-1 binding elements at positions -217 (HRE1) and +208 (HRE2) relative to the transcription start site (B). Appropriate controls were performed by transfection of the "empty" pGL2basic vector. In some experiments, a HIF-1 expression construct was cotransfected into U-2OS cells at normoxia (C, D). Luciferase activities were normalized to ß-galactosidase in each sample. Site-directed mutagenesis of HRE1 but not of HRE2 resulted in a loss of sensitivity of the Wt1 promoter to CoCl2 and HIF-1 (D). Values shown are means ± SE of 5 independent experiments, each performed in duplicate. *P < 0.05 was considered statistically significant (ANOVA with Bonferroni test as post hoc test).

5. A HIF-1 binding site in the Wt1 promoter confers responsiveness to hypoxic stimuli
To explore whether the promoter of the Wt1 gene confers hypoxia sensitivity to a heterologous reporter, a 767 bp sequence extending from -513 to +254 nucleotides relative to the transcription start site in the mouse Wt1 gene was ligated into the pGL2basic luciferase reporter plasmid and transiently transfected into U-2OS cells (Fig. 2B ). Normalized luciferase activities were enhanced sixfold in hypoxic (1% O₂) vs. normoxic (20% O₂) cells (Fig. 2C ). Treatment of the transfected cells with CoCl₂ (100 µM) and cotransfection of a HIF-1 expression construct increased reporter activities 9- and 21-fold, respectively (Fig. 2C ). Sequence analysis revealed two potential HIF-1 consensus binding elements (5'-RCGTG-3') at positions -217 (HRE1) and +208 (HRE2) of the transfected Wt1 DNA (Fig. 2B ). Site-directed mutagenesis of HRE1 but not of HRE2 abrogated the sensitivity of the Wt1 promoter to CoCl₂ and HIF-1 (Fig. 2B ). Binding of nuclear extracts from hypoxic and CoCl₂-treated U-2OS cells to HRE1 was demonstrated by EMSA. Binding activity in the nuclear extracts could be competed with an unlabeled oligonucleotide carrying the hypoxia-responsive 3' enhancer from the human erythropoietin gene. Preincubation with anti-HIF-1 antibody supershifted the retardation band, indicating that the binding protein in nuclear extracts from hypoxic cells was HIF-1.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate in this study that the Wilms’ tumor gene Wt1 is regulated in an oxygen-dependent manner in rat kidney and heart. Stimulation of Wt1 at low oxygen tension involves hypoxia-inducible factor-1 (HIF-1), based on the following. First, the promoter of the Wt1 gene confers sensitivity to hypoxia and "hypoxia-like" stimuli (CoCl₂, DFX). Second, deletion of a hypoxia-responsive consensus element (HRE) from the proximal Wt1 promoter abrogates induction by HIF-1. Finally, the hypoxia-sensitive sequence in the Wt1 promoter binds to nuclear protein from hypoxic cells. On the basis of competition and supershift experiments, we conclude that the binding protein in the hypoxic nuclear extracts is HIF-1.

Induction of Wt1 at low oxygen tension could become relevant with regard to the dual role of Wt1 in tumor growth and organ development. Wilms’ tumor (nephroblastoma) is thought to arise from the condensed metanephric blastema, which continues to proliferate rather than differentiating into epithelial components of the mature nephron. The pediatric tumors presumably develop from persistent foci of pluripotent renal cells, the so-called nephrogenic rests. Loss-of-function mutations in the Wt1 gene are responsible for 15% of sporadic Wilms’ tumors. Remarkably, Wt1 is highly expressed in a significant percentage of these tumors, suggesting a more complex role in tumorigenesis than predicted by Knudson in his two-hit model of a tumor suppressor. Our findings raise the possibility that local tissue hypoxia may contribute to enhanced Wt1 expression in Wilms’ tumors through activation of HIF-1. Consistently, HIF-1 and vascular endothelial growth factor were coexpressed in the majority of Wilms’ tumors independent of the histological subtype.

Mouse embryos with disrupted Wt1 gene exhibit lack of normal metanephric kidney development and die in utero before end-gestation. Histomorphological analyses of metanephric kidneys revealed that the ureteric bud fails to grow out from the Wolffian duct and that cells of the metanephric mesenchyme become apoptotic in Wt1^-/- embryos.

Wt1 is a transcriptional activator of the Bcl-2 gene that encodes an anti-apoptotic protein. Absence of Wt1 from the metanephric blastema of homozygous null mutants may increase the susceptibility of these cells to proapoptotic stimuli. Tubular Wt1 immunostaining in hypoxic kidneys was associated with activation of Bcl-2 expression, a decrease in the number of TUNEL-positive cells, and reduced caspase-3/7 activity. These observations raise the interesting possibility that induction of Bcl-2 in hypoxic kidneys involves Wt1 and may protect cells from apoptosis. Consistently, hypoxic preconditioning reduced cell death by ischemia/reperfusion injury in rat kidney. It remains to be clarified whether Wt1 can directly activate Bcl-2 in the kidneys of hypoxic rats. The intense cytoplasmic labeling of renal tubules suggests that Wt1 may exert its effects in these cells through mechanisms in addition to transcriptional regulation.

In summary, our findings demonstrate that hypoxia is a stimulus for Wt1 in the kidneys and heart. Besides other yet unidentified mechanisms, Wt1 expression at reduced oxygen tension involves activation of the promoter of the Wt1 gene by HIF-1.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram .Hypoxia increases HIF-1, which activates Wt1 expression through binding to a hypoxia-responsive element (HRE) in the proximal Wt1 promoter. The Wt1 zinc finger protein in turn may regulate a variety of genes including the anti-apoptotic molecule Bcl-2. At normoxia, HIF-1 is subjected to proteosomal degradation upon oxygen-dependent hydroxylation of two proline residues.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1065fje; doi: 10.1096/fj.02-1065fje

² K.-D.W. and N.W. contributed equally to this work.

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