Amine Oxidase Copper-containing 1 (AOC1) Is a Downstream Target Gene of the Wilms Tumor Protein, WT1, during Kidney Development*

Background: Polyamines and their diamine precursor putrescine are ubiquitous organic polycations involved in cell growth and proliferation. Results: The Wilms tumor suppressor, WT1, stimulates transcription of the AOC1 gene, which encodes the key enzyme for putrescine breakdown. Conclusion: WT1-dependent regulation of putrescine degradation, mediated by AOC1, has a role in kidney morphogenesis. Significance: The findings provide novel insights into transcriptional mechanisms controlling genitourinary development. Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Polyamines are low molecular weight organic polycations that are found in the cells of all living organisms (1). Due to their positive charges, polyamines can bind to macromolecules such as RNA, DNA, and proteins (2,3). The natural polyamines spermidine and spermine and their diamine precursor putrescine regulate a variety of physiological processes including gene expression, membrane stabilization, ion channel gating, and cell proliferation (1,4). Polyamines have been implicated in various pathologies, e.g. stroke, tumor expansion, inflammation, and ischemic tissue injury (5)(6)(7)(8)(9). Consistent with their roles in cell growth and survival, high amounts of polyamines have been detected in proliferating cells during development but also in mature tissues with extensive protein synthesis (1).
The rate-limiting step in polyamine biosynthesis is the conversion of the amino acid ornithine to the diamine precursor putrescine, which is catalyzed by ornithine decarboxylase 1 (ODC1). 2 Addition of two aminopropyl groups to putrescine in a two-step reaction produces spermidine and spermine, respectively (10,11). Recent findings indicate that the availability of polyamines is determined not only by the rate of synthesis but also by the activity of the degradation pathways. Although spermine and spermidine are degraded by the consecutive action of spermidine/spermine N 1 -acetyltransferase 1 and polyamine oxidase, putrescine is the preferred substrate for breakdown by amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) (12,13).
AOC1 is a homodimeric glycoprotein with an apparent molecular mass of 186 kDa that was initially thought to constitute a component of the amiloride-sensitive sodium channel (14,15). Subsequent studies revealed that AOC1 is indeed a secreted diamine oxidase, which deaminates putrescine and histamine, thereby generating hydrogen peroxide (16,17). Enzymatic activity of AOC1 can be inhibited by amiloride and some of its derivatives such as aminoguanidine (18,19). AOC1 is strongly expressed in the kidneys (20,21), placenta (22), intestine (23,24), and lungs with lower levels in the brain (21,25). Very little is known about the molecular mechanisms regulating AOC1 gene expression. The proximal AOC1 promoter * This work was supported by Deutsche Forschungsgemeinschaft Grant region is GC-rich and displays no TATA box, CAAT box, or consensus initiator sequence (16). Liang et al. (26) reported that estrogen can induce AOC1 expression via CCAAT/enhancerbinding protein-␤ in mouse uterus during embryo implantation and decidualization. Other putative trans-acting factors include cyclic AMP, bone morphogenetic protein 2, and ERK1/2 (26).
Recent findings indicate that polyamines are involved in normal kidney development (27). Nephrogenesis is accomplished by the reciprocal interaction between two tissues: the epithelial ureteric bud, which is formed from the nephric duct, and the surrounding metanephric mesenchyme (28). Signals derived from the metanephric blastema stimulate the dichotomous branching of the invading ureteric bud, which in turn induces the mesenchymal cells to condense, aggregate, and undergo nephron differentiation (28). Hence, kidney morphogenesis relies on a mesenchymal-to-epithelial transition that is regulated by various transcription factors and secreted molecules.
Interestingly, ODC1 inhibition in ex vivo cultured murine embryonic kidneys resulted in a smaller organ size, fewer epithelial ureteric bud branches, and delayed tubule formation (27). These dysmorphologies were associated with reduced cell proliferation and alteration in mesenchymal gene expression, suggesting that polyamines are important for normal murine kidney development (27). ODC1 expression is controlled on the molecular level by the transcription factor WT1, which can either repress or activate the proximal ODC1 promoter depending on the cellular context (29). Mice with homozygous Wt1 deletion (Wt1 Ϫ/Ϫ ) are embryonic lethal and exhibit agenesis of the kidneys and gonads among other defects (30 -35). Thus, regulation of polyamine synthesis appears to be one of the mechanisms by which WT1 facilitates the formation of the genitourinary system. However, the exact role of WT1 in regulating polyamine homeostasis, in particular its degradation pathway, has not been explored yet. In view of this background, our study served a 2-fold purpose. First, it was aimed at analyzing the distribution of AOC1 in developing kidneys. Second, we examined whether expression of the gene encoding AOC1 is regulated by the Wilms tumor transcription factor, WT1. We report here that AOC1 is indeed a transcriptional downstream target gene of WT1. Importantly, AOC1 inhibition influenced branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-dependent manner. These results suggest a role for AOC1 in the formation of the murine genitourinary system.

EXPERIMENTAL PROCEDURES
Animals-Mouse breeding pairs (C57/BL6 strain) were mated in the in-house animal facility in compliance with the local laws (permit number T0308/12). The morning of vaginal plugs was considered as 0.5 day postconception (d.p.c.). Sex determination of the embryos was performed by PCR amplification of the Y chromosomal gene Kdm5d from genomic DNA using the following primers: mKdm5d-F, CTGAAGCTTTTG-GCTTTGAG; mKdm5d-R, CCACTGCCAAATTCTTTGG (36).
Plasmids-A 367-base pair (bp) DNA sequence (from Ϫ301 to ϩ66 bp relative to the transcription start site) and a 113-bp DNA sequence (from Ϫ47 to ϩ66 bp relative to the transcription start site) of the human AOC1 gene (Ensembl accession number ENSG00000002726) were cloned by PCR utilizing a bacterial artificial chromosome (imaGenes, Berlin, Germany, clone RP4-548K24) as template. The amplified product was ligated into the KpnI and NheI restriction sites of the pGL3-Basic reporter plasmid (Promega, Mannheim, Germany). The PCR primers that were used for DNA amplification are listed in Table 1. Site-directed base pair mutations were introduced in the AOC1 promoter sequence by PCR as described elsewhere (40,41). All constructs were analyzed by automated DNA sequencing (EurofinsMWG, Ebersberg, Germany). The expression construct for the murine WT1(ϪKTS) protein was kindly provided by Dr. Daniel Haber (Massachusetts General Hospital, Boston, MA).
Cell Transfections and Reporter Gene Assays-HEK293 cells were expanded to ϳ40% confluence in 24-well tissue culture plates. Firefly luciferase constructs (150 ng each) harboring the indicated sequences of the human AOC1 promoter were transiently transfected along with a WT1(ϪKTS) expression construct (150 ng) and a Renilla luciferase plasmid (50 ng) using the FuGENE 6 reagent (1 l/well) as described in the supplier's manual (Promega). As negative controls, transfections were performed with the pGL3-Basic reporter plasmid (Promega) and the empty pCB6ϩ expression vector, respectively. After 24 h, the transfected cells were lysed in Reporter Lysis Buffer (Promega), and luciferase activities were measured in a luminometer (FB 12 luminometer, Berthold Detection Systems, Pforzheim, Germany) as described (40,41). Data are presented as the ratios of firefly versus Renilla luciferase activities.
Electrophoretic Mobility Shift Assay (EMSA)-Non-radioactive EMSAs were performed with purified GST-tagged recombinant WT1 protein (42) and double-stranded oligonucleotides ( Table 2), which were selected on the basis of predicted WT1 binding sites in the proximal promoter of the human AOC1 gene. All oligonucleotides were 3Ј-digoxigenin-labeled using the DIG Oligonucleotide 3Ј-End Labeling kit (Roche Applied Science). Likewise, double-stranded oligonucleotides with mutations in the WT1 binding motifs ( Table 2) were 3Ј-digoxigenin-labeled. For competition experiments, we used an oligonucleotide with a previously identified WT1 binding sequence in the promoter of the murine Adamts16 gene (43). The binding reactions were carried out at room temperature for 1 h using 0.75 g of recombinant WT1 protein and 1 pmol of labeled oligonucleotide in 1ϫ reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl 2 , 1 mM EDTA, 5 mM DTT, 5 mM phenylmethylsulfonyl fluoride). DNA-bound proteins were separated on a non-denaturing 6% polyacrylamide gel and transferred with a semidry blotting system (Bio-Rad) on a positively charged nylon membrane (Roche Applied Science). After UV cross-linking (Stratalinker UV crosslinker, Stratagene) and treatment with blocking reagent (Roche Applied Science), the membrane was incubated with anti-3Јdigoxigenin-alkaline phosphatase antibody (diluted 1:10,000; catalogue number 11093274910, Roche Applied Science). Proteins were detected by nitro blue tetrazolium/5-bromo-4chloro-3-indolyl phosphate staining (Roche Applied Science).
Chromatin Immunoprecipitation (ChIP) Assay-M15 cells were expanded in 10-cm tissue culture plates to ϳ80% confluence and used at ϳ1 ϫ 10 6 cells per assay. After fixation with 4% formaldehyde, the cells were disrupted by ultrasonication (Labsonic U, B. Braun, Melsungen, Germany) and centrifuged. The supernatants were diluted in immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1) and precleared for 1 h at 4°C with DNA-blocked protein G-agarose (Merck Millipore). The following antibodies and control sera were used (0.6 g each) for overnight incubation at 4°C: normal rabbit IgG (catalogue number sc-2027, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal anti-WT1 antibody (catalogue number sc-192, Santa Cruz Biotechnology, Inc.). The immunoprecipitates were bound to DNA-blocked protein G-agarose during 1-h incubation at 4°C. Following several washing steps, the DNA was eluted in 1% SDS, 0.1 M NaHCO 3 ; extracted in 25:24:1 phenol/ chloroform/isoamyl alcohol according to the supplier's instructions (Invitrogen); and subsequently precipitated in 100% ethanol. Immunoprecipitated DNA was amplified by quantitative PCR using primer pairs that flanked the identified WT1 binding sites in the murine Aoc1 promoter and normalized to DNA amplified from the last intron of the murine ␤-actin gene.
Organ Culture Experiments-Kidneys and gonads were excised from embryos of timed pregnant mice (C57BL6 strain). The matched organs of each embryo were cultured separately on two polyethylene terephthalate Transwell filters with 0.4-m pore size (Corning, Inc.). This procedure allowed for a pairwise comparison between cultured explants from a single donor. The Transwell filters were kept in DMEM/nutrient mixture with stable L-glutamine supplemented with 10% FCS, 100 IU/ml penicillin (PAA Laboratories), and 100 g/ml streptomycin (PAA Laboratories) at 37°C in a humidified atmosphere with 5% CO 2 . To study branching morphogenesis, the kidneys were isolated from mouse embryos at the indicated developmental stages and cultured ex vivo as described elsewhere (43). The organ rudiments were treated with 1 g/l AOC1 protein isolated from pig kidneys (Sigma-Aldrich), 100 M putrescine (Sigma-Aldrich), or 1 M AOC1 inhibitor aminoguanidine (Sigma-Aldrich). In some experiments, 200 M L-ascorbic acid (vitamin C; Roth, Karlsruhe, Germany) was added to the culture medium of the preparations. After 72 h, the explants were formalin-fixed and double stained with FITC-conjugated antipancytokeratin antibody (diluted 1:100; Sigma-Aldrich). Photographs were taken, and branching of the pancytokeratinstained ureter was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). Knockdown of WT1 was achieved by incubation of the organ cultures for 72 h either with Wt1 antisense (CAGGTCCCGCACGTCGGAACCCAT) or mismatch (CAGCTCCGGCACCTCGCAACCGATG) vivomorpholinos at 10 M each (Gene Tools, Philomath, OR) (44). Whole cell lysates and total RNA were prepared from cultured organs and cells as described below.
Preparation of RNA and Reverse Transcription (RT)-Quantitative PCR-Total RNA was isolated from tissues and primary cells with the RNeasy Micro kit (Qiagen, Hilden, Germany) and from permanent cell lines with the TRIzol LS reagent (Invitrogen). First strand cDNA synthesis was carried out using oligo-(dT) primers and Superscript TM II reverse transcriptase (Invitrogen). Real time PCR amplification was performed with SYBR Green PCR Master Mix and the StepOnePlus TM system (Invitrogen) as described in detail elsewhere (45). The PCR primers for real time RT-PCR are listed in Table 1. Relative transcript levels were obtained by subtracting the threshold cycle (Ct) value of the housekeeping gene (␤-actin or Gapdh) from the corresponding threshold cycle value of the gene of interest. Differences in mRNA levels were calculated according to the expression 2 ⌬⌬Ct .
SDS-PAGE and Immunoblotting-Cells and tissues were lysed in Laemmli buffer (50 mM Tris-HCl, pH 6.8, 4 M urea, 1% (w/v) SDS, 7.5 mM DTT), disrupted by ultrasonication (Labsonic U), and subsequently heated to 95°C for 5 min. Protein concentrations were measured spectrophotometrically. Bromphenol blue (0.001%, w/v) was added, and 20 g of protein were loaded per lane and separated on a 10% denaturing poly-acrylamide gel. The proteins were transferred onto a polyvinylidene difluoride membrane (GE Healthcare) with the use of a semidry blotting apparatus (Bio-Rad). Nonspecific binding activity was reduced by incubating the membranes for 60 min at room temperature in 5% nonfat milk (Roth) in TBS, 0.1% Tween 20. Incubation with WT1 antibody (diluted 1:400; C19, catalogue number sc-192, Santa Cruz Biotechnology, Inc.) in 2.5% nonfat milk (Roth) in TBS/Tween 20 was performed overnight at 4°C. After washing with TBS/Tween 20, the antibodies were detected with a peroxidase-coupled IgG (diluted 1:20,000; donkey anti-rabbit IgG-HRP, catalogue number sc-2313, Santa Cruz Biotechnology, Inc.), and the reaction products were visualized with Western Lightning TM Plus ECL reagents (PerkinElmer Life Sciences) following the user's manual. Equal protein loading was assessed with an actin antibody (diluted 1:6,000; anti-actin clone C4, catalogue number MAB1501R, Millipore) after stripping the membranes with 1:5 diluted 1 N NaOH in distilled water for 10 min.
Statistics-Two-tailed Student's t test (paired and unpaired) and analysis of variance with Tukey's post hoc test were performed as indicated to reveal statistical significances. p values less than 0.05 were considered significant.

AOC1 and WT1 Proteins Are Co-expressed in Embryonic
Kidneys-Although AOC1 protein can be isolated from the kidneys of various species (20,21), little is known about its cellular localization and function during embryonic development. We used a polyclonal rabbit anti-AOC1 antibody to visualize AOC1-expressing cells in developing kidneys. Anti-WT1 antibodies raised in rabbit and mouse were used to detect WT1 protein in tissue sections of mice and rats, respectively. Single immunostainings of AOC1 and WT1 on consecutive slices of mouse embryos (13.5 d.p.c.) identified both proteins in the developing kidney, testis, and mesonephros ( Fig. 1, a and b). AOC1 immunoreactivity was also visible in the adrenal gland (Fig. 1a). No fluorescence signal was obtained when normal rabbit serum instead of primary antibodies was used (Fig. 1c). Double immunostainings on tissue sections of rat embryos (16.5 d.p.c.) established an overlapping distribution of AOC1 and WT1 proteins in the glomerular precursors of the developing kidney (Fig. 1, d-g). Additional AOC1 immunoreactivity was detected in renal cells, which did not contain WT1 (Fig. 1,  d, e, and g).
Knockdown of WT1 in Embryonic Kidneys and Gonads Reduces AOC1 Expression-Co-expression of AOC1 and WT1 suggests a regulatory link between both proteins in different cell types of the genitourinary system (Fig. 1). We therefore addressed the question of whether WT1 is indeed necessary for normal expression of Aoc1 in embryonic kidneys and gonads. To this end, WT1 protein translation was inhibited in cultured murine embryonic kidneys utilizing a sequence-specific Wt1 vivo-morpholino (Wt1 morpholino) (Fig. 2). Incubation of the contralateral kidney with a Wt1 mismatch vivo-morpholino (mismatch morpholino) containing five non-matching nucleotides served as a negative control. Aoc1 mRNA expression was analyzed by RT-real time PCR after 72 h of culture in the presence of either Wt1 or mismatch vivo-morpholino. Knockdown of WT1 significantly reduced Aoc1 mRNA levels in the kidneys at 11.5, 12.5, and 13.5 d.p.c. by 70.8 Ϯ 32.2, 51.9 Ϯ 38.5, and 33.5 Ϯ 22.5%, respectively ( Fig. 2A). Knockdown efficiencies were assessed by immunoblotting with anti-WT1 antibody. Representative immunoblots are shown below the bar graphs (Fig. 2C). Interestingly, mRNA levels of Odc1, a known target gene of WT1 (29,46), were reduced by 26.1 Ϯ 37.6 (11.5 d.p.c., n ϭ 11) and 30.0 Ϯ 29.0% (12.5 d.p.c., n ϭ 12) in kidneys treated with Wt1 morpholino (p Ͻ 0.05, paired t test). No significant changes in Odc1 transcripts were observed between Wt1 morpholino-and mismatch morpholino-treated kidneys at 13.5 d.p.c. (n ϭ 6) (Fig. 2B). Antisense inhibition of WT1 was associated with a significant reduction of intrarenal ureter branching at different developmental stages (Fig. 2D). In line with the findings made on embryonic kidneys, WT1 was required for normal Aoc1 mRNA levels also in the developing gonads of male and female mice. Thus, WT1 knockdown reduced Aoc1 transcripts in the testis by 72.6 Ϯ 25.871.4 Ϯ 21.8, and 30.3 Ϯ 66.0% at 11.5, 12.5, and 13.5 d.p.c., respectively (Fig.  3A), and in the ovary by 65.3 Ϯ 32.6 (11.5 d.p.c.), 57.0 Ϯ 41.1 (12.5 d.p.c.), and 41.4 Ϯ 51.8% (13.5 d.p.c.) (Fig. 3B). These findings indicate that WT1 is necessary for normal expression of Aoc1 in murine embryonic kidneys and gonads, particularly during the early developmental stages.
Aoc1 mRNA Levels Correlate with WT1 Protein in Permanent Cell Lines-The murine mesonephros-derived cell line M15 was used to explore whether Aoc1 transcript levels are affected by changes in cellular WT1 protein content. For this purpose, endogenous WT1 expression was inhibited by transfection of M15 cells with a pool of four different sequence-specific Wt1 siRNAs. Pooled non-targeting siRNAs were transfected as controls. Aoc1 transcripts were determined by RT-real time PCR, and knockdown efficiency was assessed by immunoblotting with anti-WT1 antibody. Silencing of Wt1 in M15 cells significantly reduced Aoc1 mRNA levels by 63.1 Ϯ 28.4% (p Ͻ 0.05 t test) (Fig. 4A). Importantly, the primary Wt1 transcript is sub-ject to alternative splicing. The usage of an alternative splice donor site at the end of exon 9 leads to the insertion of three additional amino acids, lysine, threonine, and serine (KTS), between zinc fingers 3 and 4 of the WT1 protein (47). Structural and functional data demonstrate that WT1(ϪKTS) proteins, which lack the tripeptide, act as transcription factors (48,49). Compared with the WT1(ϪKTS) isoforms, WT1 proteins with the KTS insertion exhibit increased RNA binding affinity and have a presumed role in mRNA processing (50,51). To distinguish whether the AOC1 gene is regulated predominantly by the WT1(ϪKTS) protein or by the WT1(ϩKTS) splice variant, we made use of cell lines with exclusive expression of either WT1 isoform. The UB27 and UD28 cell lines contain the WT1(ϪKTS) and the WT1(ϩKTS) splice variants, respectively, under the control of a tetracycline-sensitive promoter (37). Removal of tetracycline from the culture medium resulted in a comparable increase of WT1 protein in both cell lines (Fig.  4D). Notably, AOC1 transcripts were elevated 3.4 Ϯ 1.5-and 3.4 Ϯ 1.1-fold at 48 and 72 h after induction of WT(ϪKTS) in UB27 cells (F(3,10) ϭ 8.195, p Ͻ 0.05, analysis of variance) (Fig.  4B). However, no significant changes in AOC1 mRNA levels were measured in response to induction of WT1(ϩKTS) in UD28 cells (Fig. 4C). These data demonstrate that AOC1 expression is stimulated by the WT1(ϪKTS) protein.
The WT1(ϪKTS) Isoform Activates the AOC1 Promoter-To determine whether WT1(ϪKTS) protein stimulates the AOC1 promoter directly, a luciferase reporter vector was cloned and co-transfected in HEK293 cells along with a WT1(ϪKTS) expression plasmid (Fig. 5A). Sequence analysis predicted a high affinity WT1 binding site in the proximal promoter of the AOC1 gene that was conserved among human and mouse. This element was located between Ϫ21 and Ϫ1 bp relative to the transcription start site (ϩ1) in the human AOC1 promoter. Three different human AOC1 promoter reporter constructs were generated. One construct, pAOC1prom-301, contained the presumed WT1 binding site in a sequence extending from FIGURE 1. AOC1 and WT1 protein expression in embryonic kidneys and gonads. Serial cryosections of a mouse embryo (13.5 d.p.c.) were stained with antibodies against AOC1 (a) and WT1 (b), respectively. Normal rabbit serum (NRbS) was used as a negative control (c). K, kidney; T, testis; M, mesonephros; A, adrenal gland. Representative double immunolabeling of AOC1 and WT1 in a 16.5 d.p.c. embryonic rat kidney (d-g) is shown. AOC1 (red) and WT1 (green) proteins were visualized with Cy3-and Alexa Fluor 488-conjugated secondary antibodies, respectively. Co-localization of AOC1 and WT1 in cells of the developing glomeruli is indicated by the yellow fluorescence signal in the merged image (g). Note that AOC1 protein is also present in cells that do not contain WT1 (d and g).
Ϫ301 to ϩ66 bp relative to the transcription start site. The second construct, pAOC1prom-47, spanned from Ϫ47 to ϩ66 bp, and the third reporter vector, pAOC1prom-301mut, contained the 367-bp insert with a mutated WT1 binding motif. Transfection of the empty pGL3-Basic reporter plasmid served as a negative control. The two constructs, pAOC1prom-301 and pAOC1prom-47, were stimulated 2.6 Ϯ 0.7-and 3.3 Ϯ 1.2-fold (p Ͻ 0.05 t test), respectively, by co-transfection of the WT1(ϪKTS) expression vector (Fig. 5A). In contrast, WT1(ϪKTS) exerted no significant effect on pAOCp1prom-301mut in which the predicted WT1 binding site had been mutated (Fig. 5A). An electrophoretic mobility shift assay was performed to investigate whether WT1(ϪKTS) protein bound to this identified element (Fig. 5B). For that purpose, recombinant WT1(ϪKTS) protein was incubated with a digoxigeninlabeled double-stranded DNA oligonucleotide carrying the predicted WT1 binding site in the AOC1 promoter (Fig. 5B,  lane 2). Binding of WT1(ϪKTS) to this oligonucleotide resulted in a band shift (arrow) that was eliminated by addition of a non-labeled competitor (Fig. 5B, lanes 3-5). The competitor consisted of an oligonucleotide (used at a 10-, 50-and 100fold molar excess) that contained the previously identified WT1 binding element of the murine Adamts16 promoter (43). Binding of Wt1(ϪKTS) protein to the AOC1 promoter oligonucleotide was abolished upon introducing single base pair mutations (Fig. 5B, lane 7). These results demonstrate that WT1(ϪKTS) protein stimulates the promoter of the AOC1 gene by interacting with a specific cis-regulatory element. ChIP was carried out to explore whether WT1 protein binds to the promoter of the Aoc1 gene in a natural chromosomal context (Fig. 5C). To this end, the chromatin of M15 cells was incubated either with anti-WT1 antibody or with normal rabbit serum. The amount of precipitated DNA was determined by real time PCR using specific primers for amplification of the murine Aoc1 promoter. Compared with the use of normal rabbit serum, Both kidneys were isolated from murine embryos at 11.5, 12.5, and 13.5 d.p.c. and cultured for 72 h in the presence of either Wt1 antisense (Wt1 morpholino) or mismatch vivo-morpholino (mismatch). Aoc1 and Odc1 transcripts were measured by real time RT-PCR and normalized to Gapdh mRNA. A pairwise comparison was performed between kidneys incubated with Wt1 morpholino and mismatch morpholino. Values of the mismatch morpholino-treated kidneys were set to 1. Significant differences in Aoc1 (A) and Odc1 (B) mRNA levels are indicated (*, p Ͻ 0.05; **, p Ͻ 0.005; Student's paired t test). Knockdown efficiencies were assessed by immunoblotting with anti-WT1 antibody using actin as a loading control (C). n.s., not significant. D, analysis of ureter branching in kidneys (11.5, 12.5, and 13.5 d.p.c.) treated with either Wt1 or mismatch vivo-morpholino (10 M each). After 48 h of culture in the presence of vivo-morpholinos, the whole mount preparations were stained with a FITCconjugated anti-pancytokeratin antibody (dark field images), and a skeleton of the ureter was extracted manually (schematic images). Branching of the ureter was analyzed from the schematic images. Data are presented as means Ϯ S.D. (error bars). * (p Ͻ 0.05) and ** (p Ͻ 0.005) indicate significant differences (Student's paired t test). incubation with anti-WT1 antibody resulted in a significant increase of Aoc1 promoter DNA (2.1 Ϯ 1.2-fold, p Ͻ 0.05, t test). Enrichment (1.7 Ϯ 0.7-fold, p Ͻ 0.05, t test) of the Amhr2 promoter, a known target gene of WT1 (52), was used as a positive control.
AOC1 Affects Branching Morphogenesis in Cultured Murine Embryonic Kidneys-The potential role of AOC1 in kidney morphogenesis was studied with murine organ explants. For this purpose, the kidneys were excised from mouse embryos of stages 11.5, 12.5, and 13.5 d.p.c. and incubated ex vivo in the presence of AOC1 (50 milliunits/ml) from porcine kidney. For comparison, 1 unit of AOC1 catalyzes 1 mol of putrescine in 1 h at 37°C. Equimolar amounts of bovine serum albumin (BSA) served as a control (Fig. 6A). Branching of the intrarenal ureter was visualized with a FITC-conjugated anti-pancytokeratin antibody. Addition of AOC1 significantly reduced the numbers of ureter branches during the developmental stages 11.5, 12.5 and 13.5 d.p.c. by 16.4 Ϯ 22.8, 10.2 Ϯ 9.5, and 20.7 Ϯ 11.0%, respectively. The lower branching density in the center of the kidney was characteristic for all AOC1-treated preparations at 13.5 d.p.c. (Fig. 6A). Notably, degradation of polyamines by AOC1 is expected to increase H 2 O 2 production (12,13). Alteration of ureteric bud branching in response to AOC1 treatment might therefore be due to a critical decrease of polyamines and/or accumulation of cytotoxic H 2 O 2 . To distinguish between these possibilities, we added AOC1 together with putrescine to the organ cultures. Combined treatment of the tissue specimens with AOC1 (50 milliunits/ml) and putrescine (100 M) further reduced (p Ͻ 0.5, paired t test) ureter branching at 11.5 (Ϫ31.1 Ϯ 25.8%, n ϭ 6), 12.5 (Ϫ30.0 Ϯ 12.9%, n ϭ 5), and 13.5 d.p.c. (Ϫ37.3 Ϯ 19.1%, n ϭ 8) (Fig. 6B). Otherwise, the free radical scavenger vitamin C (200 M) prevented the inhibition of ureteric bud branching that was caused by AOC1 (Fig.  6C). However, combined treatment with AOC1 and vitamin C significantly increased the number of intrarenal branching points at 13.5 d.p.c. (Fig. 6C). We therefore assume that impaired ureter growth in response to AOC1 treatment resulted from H 2 O 2 cytotoxicity rather than polyamine depletion. Addition of putrescine (100 M) alone inhibited ureter branching at 13.5 d.p.c. by 15.5 Ϯ 21.2% but had no significant effect during earlier developmental stages (11.5 and 12.5 d.p.c.) (Fig. 7A). Aminoguanidine was used to inhibit endogenous AOC1 activity in kidney organ cultures. Incubation with aminoguanidine (1 M) significantly increased intrarenal ureter branching by 25.7 Ϯ 31.4% at 11.5 d.p.c. but reduced the number of branches by 10.1 Ϯ 15.3% (p Ͻ 0.05) at 13.5 d.p.c. (Fig.  7B). Aminoguanidine had no significant effect on ureteric bud branching at 12.5 d.p.c. (Fig. 7B). These findings indicate that branching morphogenesis of the kidney is influenced by polyamines in a developmental stage-specific manner.

DISCUSSION
The mammalian kidney develops through an intricate process of branching morphogenesis, which is initiated by the outgrowth of the ureter from the nephric duct and subsequent invasion into the metanephric mesenchyme (28). During this course, signals emanating from the branching ureteric bud tips induce the mesenchymal cells to aggregate and epithelialize. Epithelial differentiation of the metanephric blastema in turn is a prerequisite for sustained ureter branching and nephron formation (28).
The Wilms tumor protein, WT1, is initially expressed in the condensing mesenchyme of the metanephric kidney and is necessary for the dichotomous growth of the ureteric bud. In the absence of WT1, the cells of the metanephric blastema become apoptotic, the ureteric bud fails to protrude into the surrounding mesenchyme, and epithelialization does not occur (30). In vitro organ culture experiments proved a requirement of WT1 not only for the early inductive events but also during the later phases of kidney formation, i.e. the differentiation and maturation of nephrons (53). In agreement with a previous investigation (44), we observed a significant reduction in the number of intrarenal ureter branching points upon Wt1 morpholino treatment at different developmental stages (Fig. 2D). These current findings suggest that transcriptional activation of the Aoc1 gene by WT1 contributes to kidney morphogenesis by regulating putrescine breakdown.
Consistently, administration of external AOC1 significantly reduced ureteric bud branching in murine embryonic kidney explants at 11.5 d.p.c., whereas inhibition of endogenous AOC1 with aminoguanidine stimulated in vitro nephrogenesis at this developmental stage (Figs. 6 and 7). In line with our observation, inhibition of ODC1, the rate-limiting enzyme for polyamine synthesis, has been shown previously to reduce cell proliferation and tubule formation in murine kidney explants at E11.0 (27). The exact mechanism how polyamines influence kidney development is unknown but may involve the regulation of genes acting on the mesenchymalepithelial balance (27).
Interestingly, aminoguanidine had no significant effect on branching morphogenesis at 12.5 d.p.c. and even inhibited tubule formation at 13.5 d.p.c. (Fig. 7B). These findings suggest that the requirement of polyamines for kidney organogenesis is developmental stage-specific. Initially, down-regulation of polyamine breakdown may promote kidney growth presumably by stimulating cell proliferation (27). As kidney formation proceeds, nephron differentiation is favored by a decrease in cell proliferation, and high polyamine levels would thus inhibit branching morphogenesis (53).
It is important to note that aminoguanidine also reduces the activities of other enzymes such as polyamine oxidase, which degrades spermine and spermidine (54), and NO synthase, which is necessary for NO production. However, the K i values for inhibition of polyamine oxidase (K i value of 70 M) and the constitutive (K i value of 0.83 mM) and inducible (K i value of 16 M) NO synthase are far above the K i value for AOC1 inhibition Interaction of WT1(ϪKTS) protein with the AOC1 promoter oligonucleotide could be competed with excess amounts of unlabeled DNA (lanes 3-5) containing the previously identified WT1 binding element of the murine Adamts16 promoter (43). Mutation of the WT1 consensus motif abrogated WT1(ϪKTS) binding (lanes 6 and 7). Interaction of WT1(ϪKTS) protein with the Adamts16 promoter element (43) served as an internal quality control (lanes 8 and 9). C, ChIP was performed to detect WT1 protein bound to the promoter region of the murine Aoc1 gene in its native chromosomal configuration. A specific antibody against WT1 was used for immunoprecipitation of chromatin prepared from M15 cells. Incubation with normal rabbit IgG (NRb-IgG) served as a negative control. Amplicons encompassing the 5Ј-flanking region of the Aoc1 gene were generated by real time PCR. The gene encoding anti-Müllerian hormone receptor 2 (Amhr2), a previously identified WT1 target (52), was used as a positive control. Data shown are means Ϯ S.D. (error bars). Statistical significances between normal rabbit IgG and anti-WT1 are indicated by an asterisk (p Ͻ 0.05, paired t test, n ϭ 7). FIGURE 6. Effect of AOC1, given either alone or in combination with putrescine and vitamin C, respectively, on branching morphogenesis in embryonic kidney explants. Kidneys were excised from murine embryos at the indicated gestational stages and incubated for 48 h in the presence of AOC1 (50 milliunits/ml) alone (A) or together with 100 M putrescine (B) and 200 M vitamin C (VitC) (C), respectively. As a control, the contralateral kidney of each embryo was treated with BSA. After 48 h of culture, the ureter was stained with a FITC-conjugated anti-pancytokeratin antibody (dark field images), and a skeleton of the ureteric bud branches was drawn manually (schematic images). Branching of the ureter was analyzed based on the schematic sketches. The absolute numbers of branches are indicated in the columns. Data are presented as means Ϯ S.D. (error bars). * (p Ͻ 0.05) and ** (p Ͻ 0.005) indicate significant differences (paired t test).
(K i value of 50 nM) (54 -56). We therefore assume that the treatment of kidney organ cultures with aminoguanidine at a 1 M concentration was effective primarily through inhibition of AOC1 activity and thus putrescine breakdown.
AOC1 does not only inactivate putrescine but also catalyzes histamine degradation (57). Histamine is produced by the histamine decarboxylase, which is expressed in the developing kidney earliest at 14.5 d.p.c. (58). Changes in histamine metabolism are therefore unlikely to account for the observed effects of AOC1 on ureteric bud branching before this time. It is also noteworthy that degradation of putrescine by AOC1 generates H 2 O 2 , which might exert toxic effects in embryonic kidney explants. In fact, inhibition of ureteric bud branching in response to external AOC1 was augmented by the simultaneous administration of putrescine (Fig. 6B). Furthermore, the free radical scavenger vitamin C prevented the inhibitory effect of AOC1 on ureteric branching at 11.5 and 12.5 d.p.c., and in the presence of vitamin C, AOC1 even increased the number of branching points at 13.5 d.p.c. (Fig. 6C). These findings suggest that the detrimental effect of AOC1 was, at least in part, due to the production of cytotoxic H 2 O 2 in addition to the depletion of putrescine (59). Hence, our data support the idea that the breakdown of polyamines is beneficial to kidney morphogenesis provided that the concomitant generation of harmful H 2 O 2 is compensated by scavenger molecules. WT1 has previously been reported to regulate the expression of ODC1, the key enzyme for polyamine synthesis, in a rather multifaceted manner. Although overexpression of WT1 in baby hamster kidney cells resulted in a decline of cellular Odc1 mRNA levels (46), WT1 enhanced ODC1 promoter activity in SV40-transfected HepG2 cells and repressed activity of the ODC1 promoter in several other cell lines (29). These findings suggest that the effect of WT1 on ODC1 expression is complex and cell type-specific. We observed a significant decrease of Odc1 transcripts in renal organ cultures upon antisense inhibition of Wt1, indicating that WT1 stimulates Odc1 expression in the developing kidney (Fig. 2).
Considering the antagonistic functions of ODC1 and AOC1 in polyamine metabolism, it seems puzzling at first glance that WT1 up-regulates both genes in embryonic kidneys (Fig. 2). In this regard, one has to keep in mind that distinct cell populations in embryonic kidneys may exhibit a specific and variable demand for polyamines during successive phases of differentiation. High levels of Odc1 mRNA were detected in ureteric bud cells, suggesting that ureter branching requires active polyamine synthesis (27). Importantly, polyamines and their diamine precursor, putrescine, are secreted molecules that can be taken up by neighboring cells through membrane transporters (60). Thus, a rise of polyamines in the metanephric mesenchyme due to secretion from the invading ureteric bud tips might be detrimental to nephron differentiation. In agreement with this possibility, incubation of murine embryonic kidney explants in the presence of putrescine significantly reduced ureter branching at 13.5 d.p.c. (Fig. 7A). Hence, stimulation of Aoc1 expression by WT1 may fulfill the purpose of fine-tuning polyamine levels in a spatial and temporal manner. This idea is supported by the co-expression of WT1 and AOC1 proteins in developing glomeruli of embryonic kidneys (Fig. 1d).
Stimulation of Aoc1 expression by WT1 may also become important for the development and function of the gonads. The mammalian gonads are formed by tissue derived from the intermediate mesoderm on either side of the embryonic midline axis (61). Development of the bipotential gonadal ridge occurs in a non-sex-specific manner in XX and XY individuals and is completed by 12 d.p.c. in rats and mice (62). WT1 acts on multiple steps during gonad development. Thus, gonadal ridge formation in Wt1-deficient mice is initiated in both males and females but then degenerates due to apoptosis, leading to gonadal agenesis (30). Polyamines are essential to male and female reproductive processes. In male reproduction, polyamines correlate with stages of spermatogenesis and regulate sperm cell motility (63). In female mammals, polyamines are involved in ovarian follicle development and ovulation (63). Recent findings have demonstrated that these processes in the male and female reproductive system also require WT1 (64,65). Thus, it appears plausible that the effects of WT1 in the gonads of both sexes are mediated to some extent by the control of polyamine homeostasis.
In summary, normal formation of the genitourinary system requires a precise adjustment of polyamines during different developmental stages. Previous studies have shown that this can be accomplished by a tight regulation of ODC1 activity (66). Our data suggest that WT1-dependent stimulation of Aoc1 gene expression contributes to the harmonization of polyamine levels in embryonic kidneys with the actual needs. Down-regulation of AOC1 due to inactivation of Wt1 is expected to disturb FIGURE 7. Effect of putrescine and AOC1 inhibition by aminoguanidine on branching morphogenesis in embryonic kidney explants. Embryonic kidney organ cultures (11.5, 12.5, and 13.5 d.p.c.) were treated for 48 h with 100 M putrescine (A) or 1 M aminoguanidine to inhibit AOC1 enzyme activity (B). The contralateral kidney of each embryo was cultured in the presence of BSA. The preparations were processed and analyzed as described in the legend of Fig. 6. The absolute numbers of branches are given in the columns. Data are means Ϯ S.D. (error bars). * (p Ͻ 0.05) and ** (p Ͻ 0.005) indicate significant differences (paired t test). the delicate balance of growth-promoting and prodifferentiating signals, thereby favoring a proliferative phenotype. Intriguingly, a significant increase in free polyamines has been reported in the peripheral blood of Wilms tumor patients (67). In light of this observation and our current findings, it might be rewarding to investigate the role of AOC1 expression and polyamine metabolism in human Wilms tumors in more detail.