Transcription Factor Wilms Research Articles

Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

Distinct global binding patterns of the Wilms tumor gene 1 (WT1) -KTS and +KTS isoforms in leukemic cells.

The zinc finger transcription factor Wilms tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (±KTS), is believed to change the DNA binding affinity of WT1, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of the WT1 -KTS isoform at the expense of the WT1 +KTS isoform is associated with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with the involvement of WT1 in acute myeloid leukemia.

The roadmap of WT1 protein expression in the human fetal heart

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

WT1 targets Gas1 to maintain nephron progenitor cells by modulating FGF signals.

Development of the metanephric kidney depends on tightly regulated interplay between self-renewal and differentiation of a nephron progenitor cell (NPC) pool. Several key factors required for the survival of NPCs have been identified, including fibroblast growth factor (FGF) signaling and the transcription factor Wilms' tumor suppressor 1 (WT1). Here, we present evidence that WT1 modulates FGF signaling by activating the expression of growth arrest-specific 1 (Gas1), a novel WT1 target gene and novel modulator of FGF signaling. We show that WT1 directly binds to a conserved DNA binding motif within the Gas1 promoter and activates Gas1 mRNA transcription in NPCs. We confirm that WT1 is required for Gas1 expression in kidneys in vivo. Loss of function of GAS1 in vivo results in hypoplastic kidneys with reduced nephron mass due to premature depletion of NPCs. Although kidney development in Gas1 knockout mice progresses normally until E15.5, NPCs show decreased rates of proliferation at this stage and are depleted as of E17.5. Lastly, we show that Gas1 is selectively required for FGF-stimulated AKT signaling in vitro. In summary, our data suggest a model in which WT1 modulates receptor tyrosine kinase signaling in NPCs by directing the expression of Gas1.

Genome-Wide Analysis of Wilms' Tumor 1-Controlled Gene Expression in Podocytes Reveals Key Regulatory Mechanisms.

The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.

WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors.

Kidney organogenesis requires the tight control of proliferation, differentiation and apoptosis of renal progenitor cells. How the balance between these cellular decisions is achieved remains elusive. The Wilms' tumour suppressor Wt1 is required for progenitor survival, but the molecular cause for renal agenesis in mutants is poorly understood. Here we demonstrate that lack of Wt1 abolishes fibroblast growth factor (FGF) and induces BMP/pSMAD signalling within the metanephric mesenchyme. Addition of recombinant FGFs or inhibition of pSMAD signalling rescues progenitor cell apoptosis induced by the loss of Wt1. We further show that recombinant BMP4, but not BMP7, induces an apoptotic response within the early kidney that can be suppressed by simultaneous addition of FGFs. These data reveal a hitherto unknown sensitivity of early renal progenitors to pSMAD signalling, establishes FGF and pSMAD signalling as antagonistic forces in early kidney development and places WT1 as a key regulator of pro-survival FGF signalling pathway genes.

Leukemia associated mutant Wilms’ tumor gene 1 protein promotes expansion of human hematopoietic progenitor cells

The transcription factor Wilms' tumor gene 1 (WT1) is highly expressed in the majority of leukemias, suggesting a role in leukemogenesis. Acquired WT1 mutations are reported as an independent predictor of poor clinical outcome, and mutations resulting in deletion of the entire DNA-binding zinc-finger domain (WT1delZ), is the most common type. The aim of this study was to study cellular effects of WT1(delZ) that may contribute to an oncogenic phenotype. We found that expression of WT1(delZ) supported proliferation of human hematopoietic CD34(+) progenitor cells. Moreover, WT1(delZ) transduced cells expressed erythroid markers, including raised levels of STAT5, independently of addition of erythropoietin. At the global gene expression level, WT1(delZ) caused upregulation of genes related to cell division and genes associated with erythroid maturation, in the absence of added erythropoietin. Our results indicate that WT1(delZ) promotes cell proliferation and expansion of progenitor cells, consistent with a possible role in leukemogenesis.

The transcription factor sry‐related HMG box‐4 (SOX4) is required for normal renal development in vivo

The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown. Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4(nephron-) mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4(nephron-) renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4(nephron-) mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5-9 months. Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo.

Abstract 775: Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro.

Abstract The transcription factor Wilms’ tumour gene 1 (WT1) is present in some primitive hematopoietic cells and is highly expressed in the majority of lymphoid and myeloid leukemias, suggesting a role for WT1 in leukemogenesis. Mutations within the WT1 gene are present in 10-15 % of patients with cytogenetically normal acute myeloid leukemias and may be an independent predictor of poor clinical outcome. The mutations are most often heterozygous frameshift mutations leading to premature termination of the reading frame and therefore deletion of the entire DNA-binding zinc-finger domain. We have investigated the cellular effects of mutant WT1 protein lacking all four zinc-fingers, WT1(delZ). We report that expression of WT1(delZ) prolongs the proliferation of human hematopoietic CD34+ progenitor cells in vitro. Moreover, WT1(delZ) transduced cells express erythroid markers, including hemoglobin and elevated STAT5 protein levels, in part independently of addition of erythropoietin. STAT5 was increased by a posttranscriptional mechanism. The erythroid phenotype was also observed at global gene expression level, where WT1(delZ) caused coordinated up-regulation of genes associated with erythroid maturation, as well as genes related to cell division, while monocytic and neutrophil genes were down modulated. Our results indicate that WT1(delZ) promotes cell proliferation and erythroid differentiation at the expense of monocytes and granulocytes. Citation Format: Karina Vidovic. Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2013-775

Role of WT1–ZNF224 interaction in the expression of apoptosis-regulating genes

The transcription factor Wilms' tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. Physical interactions of other cellular proteins with WT1 are known to modulate its function. We previously identified the Krüppel-like zinc-finger protein, ZNF224, as a novel human WT1-associating protein that enhances the transcriptional activation of the human vitamin D receptor promoter by WT1. Here, we have analyzed the effects of WT1-ZNF224 interaction on the expression of apoptosis-regulating genes in the chronic myelogenous leukemia (CML) K562 cell line. The results demonstrated that ZNF224 acts in fine tuning of WT1-dependent control of gene expression, acting as a co-activator of WT1 in the regulation of proapoptotic genes and suppressing WT1 mediated transactivation of antiapoptotitc genes. Moreover, the DNA damaging drug cytosine arabinoside (ara-C) induces expression of ZNF224 in K562 cells and this induction enhances cell apoptotic response to ara-C. These findings suggest that ZNF224 can be a mediator of DNA damage-induced apoptosis in leukemia cells.

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

Transcription of nephrin-Neph3 gene pair is synergistically activated by WT1 and NF- B and silenced by DNA methylation

Nephrin and Neph3 are homologous molecules expressed in the podocyte slit diaphragms that are essential for normal glomerular ultrafiltration. Nephrin and Neph3 genes form a bidirectional gene pair suggesting that they may share key features in their regulation. We investigated if nephrin and Neph3 genes have similar mechanisms in their transcriptional regulation focussing on transcription factor Wilms' tumour 1 (WT1) and nuclear factor-κB (NF-κB) and DNA methylation. Transcriptional regulation of nephrin and Neph3 by WT1 and NF-κB was analysed by overexpression studies, reporter gene assay and chromatin immunoprecipitation using A293 cells and cultured podocytes. The interaction between WT1 and NF-κB was studied by co-immunoprecipitation. The effect of NF-κB activator tumour necrosis factor-α (TNF-α) with or without NF-κB pathway inhibitor (BAY 11-7082) on nephrin and Neph3 messenger RNA (mRNA) expression and on cellular distribution of NF-κB was determined by quantitative polymerase chain reaction (PCR) and immunostaining, respectively. The role of DNA methylation in regulating nephrin and Neph3 genes was studied by demethylating agent (5-aza-2'-deoxycytidine) treatment and quantitative PCR. WT1 and NF-κB interact with nephrin and Neph3 promoter and cooperatively regulate nephrin and Neph3. The cooperation was further supported by the physical interaction between WT1 and NF-κB. TNF-α increased nephrin and Neph3 mRNA expression and this effect was mediated by NF-κB. Furthermore, DNA methylation played a role in silencing nephrin and Neph3 expression in a cell-type and differentiation stage-dependent manner. These results provide novel insights into the transcriptional regulation of nephrin and Neph3 genes and indicate that nephrin and Neph3 share the same mechanisms in their regulation.

WT1-Dependent Sulfatase Expression Maintains the Normal Glomerular Filtration Barrier

Paracrine signaling between podocytes and glomerular endothelial cells through vascular endothelial growth factor A (VEGFA) maintains a functional glomerular filtration barrier. Heparan sulfate proteoglycans (HSPGs), located on the cell surface or in the extracellular matrix, bind signaling molecules such as VEGFA and affect their local concentrations, but whether modulation of these moieties promotes normal crosstalk between podocytes and endothelial cells is unknown. Here, we found that the transcription factor Wilms' Tumor 1 (WT1) modulates VEGFA and FGF2 signaling by increasing the expression of the 6-O-endosulfatases Sulf1 and Sulf2, which remodel the heparan sulfate 6-O-sulfation pattern in the extracellular matrix. Mice deficient in both Sulf1 and Sulf2 developed age-dependent proteinuria as a result of ultrastructural abnormalities in podocytes and endothelial cells, a phenotype similar to that observed in children with WT1 mutations and in Wt1(+/-) mice. These kidney defects associated with a decreased distribution of VEGFA in the glomerular basement membrane and on endothelial cells. Collectively, these data suggest that WT1-dependent sulfatase expression plays a critical role in maintaining the glomerular filtration barrier by modulating the bioavailability of growth factors, thereby promoting normal crosstalk between podocytes and endothelial cells.

Abstract 2169: The effect of WT1 on E-cadherin expression in prostate cancer cells

Abstract Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. One of the genes expressed in prostate cancer epithelium is the zinc finger transcription factor Wilms’ Tumor 1, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the regulation of cell adhesion molecules such as E-cadherin in NIH 3T3 cells and epicardial cells. Loss of E-cadherin expression is frequently associated with increased cellular motility and tumor invasion. Several regulatory mechanisms controlling E-cadherin gene expression have been proposed in breast cancer cells, however the mechanisms of regulation of E-cadherin gene expression in prostate cancer cells are not yet understood. The objective of this study was to determine whether WT1 might regulate E-cadherin expression and contribute to cancer progression in prostate cancer cells. First, potential WT1 binding sites were identified in the promoter of the E-cadherin gene using a bioinformatics approach. Chromatin Immunoprecipitation (ChIP) showed direct in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells. The effect of transfection of prostate cancer cells with a GFP/WT1 expression construct was then tested by quantitative real-time PCR (QRT-PCR). QRT-PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected prostate cancer cells. Conversely, knockdown of WT1 mRNA in siWT1 transfected LNCaP cells, showed increased levels of E-cadherin mRNA. Moreover, co-transfection of WT1 expression construct with an E-cadherin promoter reporter construct showed that WT1 decreased the activity of the proximal E-cadherin promoter in PC3 cells. Overall these results demonstrated that WT1 modulated E-cadherin expression in prostate cancer cells and suggests a novel mechanism whereby WT1 may increase migration by decreasing E-cadherin levels, a novel role for WT1 mediated prostate cancer progression. This work was supported by NIHR15CA11360 and Ohio Board of Regents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2011-2169

Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models

KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

Kinetic behaviour of WT 1’s zinc finger domain in binding to the alpha-actinin-1 mRNA

The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C 2H 2-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.

Characterization of the molecular differences between ovarian endometrioid carcinoma and ovarian serous carcinoma

The histopathological diagnosis of high-grade endometrioid and serous carcinoma of the ovary is poorly reproducible under the current morphology based classification system, especially for anaplastic, high-grade tumours. The transcription factor Wilms' tumour-1 (WT1) is differentially expressed among the gynaecological epithelia from which epithelial ovarian cancers (EOCs) are believed to originate. In EOCs, WT1 protein is observed in the majority of serous carcinomas and in up to 30% of endometrioid carcinomas. It is unclear whether the latter is a reflection of the actual incidence of WT1 protein expression in endometrioid carcinomas, or whether a significant number of high-grade serous carcinomas have been misclassified as endometrioid carcinoma. Several genetic aberrations are reported to occur in EOCs. These include mutation of the TP53 gene, aberrant activation of beta-catenin signalling and loss of PTEN protein expression, among others. It is unclear whether these aberrations are histotype-specific. The aim of this study was to better define the molecular characteristics of serous and endometrioid carcinomas in an attempt to address the problems with the current histopathological classification methods. Gene expression profiles were analysed to identify reproducible gene expression phenotypes for endometrioid and serous carcinomas. Tissue microarrays (TMA) were used to assess the incidence of TP53, beta-catenin and PTEN aberrations in order to correlate their occurrence with WT1 as an immunohistochemistry based biomarker of serous histotype. It was found that nuclear WT1 protein expression can identify misclassified high-grade endometrioid carcinomas and these tumours should be reassigned to serous histotype. Although low-grade endometrioid carcinomas rarely progress to high-grade carcinomas, a combined WT1-negative, TP53-positive immunophenotype may identify an uncommon high-grade subtype of ovarian endometrioid carcinoma. GEO database: array data accession number GSE6008.

Identification of an Immunogenic HLA-A*0201-binding T-cell Epitope of the Transcription Factor PAX2

PAX2 is a transcription factor and member of the highly conserved family of paired box genes. PAX2 is aberrantly expressed in a variety of solid and hematologic malignancies. PAX2 regulates the transcription factor Wilms tumor gene 1, which is a promising target of cancer immunotherapy. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of PAX2 which could be useful for cancer immunotherapy. Thirteen potential HLA-A*0201 epitopes were predicted by a major histocompatibility complex binding algorithm (SYFPEITHI) and a proteasome cleavage algorithm (PAProC) and screened for recognition by T cells from HLA-A*02-positive cancer patients using intracellular cytokine cytometry. Epitope-specific T cells were generated from CD4CD25 regulatory T-cell-depleted peripheral blood mononuclear cell. Nine of 20 colorectal cancer patients, 1 of 13 renal cell carcinoma patients, and 2 of 17 lymphoma patients had a spontaneous CD8 T-cell response toward at least 1 of 6 PAX2 peptide pools. None of the 20 healthy subjects showed reactivity toward PAX2. PAX2.337-345 (TLPGYPPHV)-specific T cells could repeatedly be generated, which specifically lysed the PAX2 expressing colorectal tumor cell line SW480. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*0201 restricted T-cell epitope and natural ligand of the tumor antigen PAX2. Thus, PAX2 is another embryonic transcription factor, which is of potential interest as immunotherapy target antigen.

Human lymphocytes express the transcriptional regulator, Wilms tumor 1: The role of WT1 in mediating nitric oxide-dependent repression of lymphocyte proliferation

The inhibitory roles of nitric oxide (NO) in T cell proliferation have been observed and studied extensively over the last two decades. Despite efforts, the fundamental pathway by which NO exerts its inhibitory actions remains to be elucidated although recent evidence suggests that the transcription factor Wilms tumor 1 (WT1) may be important. WT1 has been linked to numerous developmental pathways in particular nephrogenesis. Due to its roles in development and cell proliferation, polymorphisms within the WT1 gene can result in malignancies such as leukemia and Wilms tumor. WT1 functions as a transcriptional regulator and its activity is controlled through phosphorylation by protein kinase A (PKA). PKA-dependent WT1 phosphorylation results in translocation of WT1 from the nucleus to the cytosol, a process that interferes with WT1 transcriptional activities. In the current study we demonstrate that WT1 is expressed in human lymphocytes. Using the proliferative compound PHA we induced T cell proliferation and growth correlated with an increase in the expression of WT1 measured by RT-PCR, flow cytometry and immunoblot. Co-stimulation with the NO donor SNOG at concentrations of 0, 100, 300 and 600 μM reduced in a concentration dependent way the PHA-induced upregulation of WT1 that correlated with a reduction in T cell proliferation. We conclude that WT1 might be an important component of the NO-dependent regulation of T lymphocyte proliferation and potential function.

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5' flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-gamma resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.