Wilms Tumor Suppressor Research Articles

Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Cardiomyocyte-Specific Wt1 Is Involved in Cardiac Metabolism and Response to Damage.

The Wilms tumor suppressor gene (Wt1) encodes a C2H2-type zinc-finger transcription factor that participates in transcriptional regulation, RNA metabolism, and protein-protein interactions. WT1 is involved in the development of several organs, including the kidneys and gonads, heart, spleen, adrenal glands, liver, diaphragm, and neuronal system. We previously provided evidence of transient WT1 expression in about 25% of cardiomyocytes of mouse embryos. Conditional deletion of Wt1 in the cardiac troponin T lineage caused abnormal cardiac development. A low expression of WT1 has also been reported in adult cardiomyocytes. Therefore, we aimed to explore its function in cardiac homeostasis and in the response to pharmacologically induced damage. Silencing of Wt1 in cultured neonatal murine cardiomyocytes provoked alterations in mitochondrial membrane potential and changes in the expression of genes related to calcium homeostasis. Ablation of WT1 in adult cardiomyocytes by crossing αMHCMerCreMer mice with homozygous WT1-floxed mice induced hypertrophy, interstitial fibrosis, altered metabolism, and mitochondrial dysfunction. In addition, conditional deletion of WT1 in adult cardiomyocytes increased doxorubicin-induced damage. These findings suggest a novel role of WT1 in myocardial physiology and protection against damage.

Adapting the pantograph limb: Differential robustness of fore- and hindlimb kinematics against genetically induced perturbation in the neural control networks and its evolutionary implications

The evolutionary transformation of limb morphology to the four-segmented pantograph of therians is among the milestones of mammalian evolution. But, it is still unknown if changes of the mechanical limb function were accompanied by corresponding changes in development and sensorimotor control. The impressive locomotor performance of mammals leaves no doubt about the high integration of pattern formation, neural control and mechanics. But, deviations from normal intra- and interlimb coordination (spatial and temporal) become evident in the presence of perturbations. We induced a perturbation in the development of the neural circuits of the spinal cord of mice (Mus musculus) using a deletion of the Wilms tumor suppressor gene Wt1 in a subpopulation of dI6 interneurons. These interneurons are assumed to participate in the intermuscular coordination within the limb and in left-right-coordination between the limbs. We describe the locomotor kinematics in mice with conditional Wt1 knockout and compare them to mice without Wt1 deletion. Unlike knockout neonates, knockout adult mice do not display severe deviations from normal (=control group) interlimb coordination, but the coordinated protraction and retraction of the limbs is altered. The forelimbs are more affected by deviations from the control than the hindlimbs. This observation appears to reflect a different degree of integration and resistance against the induced perturbation between the limbs. Interestingly, the observed effects are similar to locomotor deficits reported to arise when sensory feedback from proprioceptors or cutaneous receptors is impaired. A putative participation of Wt1 positive dI6 interneurons in sensorimotor integration is therefore considered.

Loss of S1P Lyase Expression in Human Podocytes Causes a Reduction in Nephrin Expression That Involves PKCδ Activation.

Sphingosine 1-phosphate (S1P) lyase (SPL, Sgpl1) is an ER-associated enzyme that irreversibly degrades the bioactive lipid, S1P, and thereby regulates multiple cellular functions attributed to S1P. Biallelic mutations in the human Sglp1 gene lead to a severe form of a particular steroid-resistant nephrotic syndrome, suggesting that the SPL is critically involved in maintaining the glomerular ultrafiltration barrier, which is mainly built by glomerular podocytes. In this study, we have investigated the molecular effects of SPL knockdown (kd) in human podocytes to better understand the mechanism underlying nephrotic syndrome in patients. A stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels. This cell line was further studied for changes in those podocyte-specific proteins that are known to regulate the ultrafiltration barrier. We show here that SPL-kd leads to the downregulation of the nephrin protein and mRNA expression, as well as the Wilms tumor suppressor gene 1 (WT1), which is a key transcription factor regulating nephrin expression. Mechanistically, SPL-kd resulted in increased total cellular protein kinase C (PKC) activity, while the stable downregulation of PKCδ revealed increased nephrin expression. Furthermore, the pro-inflammatory cytokine, interleukin 6 (IL-6), also reduced WT1 and nephrin expression. In addition, IL-6 caused increased PKCδ Thr505 phosphorylation, suggesting enzyme activation. Altogether, these data demonstrate that nephrin is a critical factor downregulated by the loss of SPL, which may directly cause podocyte foot process effacement as observed in mice and humans, leading to albuminuria, a hallmark of nephrotic syndrome. Furthermore, our in vitro data suggest that PKCδ could represent a new possible pharmacological target for the treatment of a nephrotic syndrome induced by SPL mutations.

Spectrum of Clinical Manifestations in Children With WT1 Mutation: Case Series and Literature Review.

BackgroundMutations of the Wilms tumor suppressor-1 gene (WT1) are associated with life-threatening glomerulopathy, disorders of sexual development, Wilm's tumor, and gonadal malignancies. Our objectives were to describe the clinical presentations, age of progression, and onset of complications of WT1 mutation through a case series and literature review.MethodsA retrospective study included all patients followed at the University of Miami/Holtz Children's Hospital from January 2000 to December 2020 with a diagnosis of WT1 mutation. A literature review of WT1 mutation cases was analyzed for clinical manifestations, karyotype, and long-term outcomes.ResultsThe WT1 mutation was identified in 9 children, median age at presentation of 0.9 years (range 1 week to 7 years). A total of four had female phenotypes, and 5 had abnormalities of male external genitalia, while all had XY karyotypes. All progressed to end-stage kidney disease (ESKD) and received a kidney transplant at a median age of 5 years (1.5–15 years). During a median time of follow-up of 9 years (range 2–28 years), there were 2 allograft losses after 7 and 10 years and no evidence of post-transplant malignancy. From 333 cases identified from the literature review, the majority had female phenotype 66% (219/333), but the predominant karyotype was XY (55%, 183/333). Of the female phenotypes, 32% (69/219) had XY sex reversal. Wilm's tumor occurred in 24%, predominantly in males with gonadal anomalies.ConclusionsEarly recognition of WT1 mutation is essential for comprehensive surveillance of potential malignancy, avoidance of immunosuppressants for glomerulopathy, and establishing long-term multidisciplinary management.

The Wilms Tumor Gene wt1a Contributes to Blood-Cerebrospinal Fluid Barrier Function in Zebrafish.

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor, which is highly conserved among vertebrates. It is a key regulator of urogenital development and homeostasis but also plays a role in other organs including the spleen and the heart. More recently additional functions for Wt1 in the mammalian central nervous system have been described. In contrast to mammals, bony fish possess two paralogous Wt1 genes, namely wt1a and wt1b. By performing detailed in situ hybridization analyses during zebrafish development, we discovered new expression domains for wt1a in the dorsal hindbrain, the caudal medulla and the spinal cord. Marker analysis identified wt1a expressing cells of the dorsal hindbrain as ependymal cells of the choroid plexus in the myelencephalic ventricle. The choroid plexus acts as a blood-cerebrospinal fluid barrier and thus is crucial for brain homeostasis. By employing wt1a mutant larvae and a dye accumulation assay with fluorescent tracers we demonstrate that Wt1a is required for proper choroid plexus formation and function. Thus, Wt1a contributes to the barrier properties of the choroid plexus in zebrafish, revealing an unexpected role for Wt1 in the zebrafish brain.

Disorders of Sex Development.

Disorders of Sex Development.

Loss of sphingosine kinase 2 enhances Wilm's tumor suppressor gene 1 and nephrin expression in podocytes and protects from streptozotocin-induced podocytopathy and albuminuria in mice

The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2-/- mice compared to STZ-treated wildtype mice.To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation.The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation.Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth.

A Unique Presentation of XY Gonadal Dysgenesis in Frasier Syndrome due to WT1 Mutation and a Literature Review.

Frasier syndrome (FS), a rare disease caused by inherited or de novo mutation in Wilm's Tumor suppressor gene 1 (WT1), is characterized by slow progressive nephropathy, XY gonadal dysgenesis (XY-DSD), and increased risk for gonadal tumors. Early childhood (1-6 years) nephropathy progresses with age to refractory nephrotic syndrome, and end-stage renal failure in late adolescence, when delayed puberty and/or primary amenorrhea are clinically evident. We report a unique case of FS presenting initially with primary amenorrhea at 16 years, without previous or concomitant renal damage. Only subsequently she developed an extremely late-onset nephropathy. Genetic analysis revealed the IVS9 + 5 G>A mutation in intron 9 of the WT1 gene. This clinical presentation and review of WT1 literature highlights the importance of considering FS in the differential diagnosis of patients with 46,XY disorders of Sexual development, even without nephropathy. Furthermore, the identification WT1 gene mutation prior to evident renal dysfunction indicates an immediate and close surveillance of renal function enabling an optimal and timely medical response.

P1818CLINICAL AND MOLECULAR CHARACTERISTICS OF CHILDREN WITH WT-1-ASSOCIATED STEROID-RESISTANT NEPHROTIC SYNDROME

Abstract Background and Aims The Wilms tumor suppressor gene 1 (WT1) (MIM 607102) has an important role in renal and gonadal development. WT1 mutations was found to be associated with a large spectrum of phenotypes with autosomal-dominant inheritance, including Wilms tumor, Denys-Drash syndrome (DDS), Frasier syndrome (FS), and isolated steroid-resistant nephrotic syndrome (SRNS). The aim of the study was to investigate clinical and molecular characteristics in children with WT1-associated SRNS. Method Retrospective analysis of phenotype and genotype of six children (3M/3F) with SRNS and heterozygous WT1 mutations was performed. Karyotypes were available in all patients. Data on renal histology had three subjects with FS. The median follow-up period was 35.5 (IQR: 20.0; 57.5) months. WT1 mutations identified by direct Sanger sequencing (n=3) and next generation sequencing (n=3). Results The median age at onset of WT1-associated SRNS was 10.0 (IQR: 5.5; 22.5) months. 4/6 (66.7%) patients presented with infantile nephrotic syndrome and 2/6 (33.3%) with SRNS. Arterial hypertension was determined in 4/6 (66.7%) of individuals. Among children with WT1-associated SRNS 3/6 (50%) patients had DDS (2M/1F) and 3/6 (50%) subjects had FS (1M/2F). Children with DDS had Wilms tumor in 2/3 (66.7%) of cases and gonadal dysgenesis with hypospadias in 1/3 (33.3%) of children. Causative mutations were identified in all three patients with DDS, including 2 earlier described c.1384C>T in exon 9 and c.1098 + 1G>A in intron 6 in WT1 gene, and one novel mutation с.1378Т>G in exon 9. All DDS children had decreased eGFR to CKD stage 2-3 in two patients aged 7 and 11 years and ESKD in one child at the age of 2 years. All 3 patients with FS had focal segmental glomerulosclerosis on biopsy. Two phenotypic girls with FS had karyotype 46, XY and presented with pseudohermaphroditism. The same earlier described splice site mutation c.1432 + 5G>A in intron 9 in WT1 gene was identified in both cases. One boy with FS had typical male chromosome pattern (46, XY) and gonadal dysgenesis. Splice site mutation c.1432 + 4C>T in intron 9 in WT1 gene was found in the child. Nobody of patients with FS developed gonadoblastoma. Progression to CKD was found in all three cases with FS, including CKD stage 2-3 in two subjects aged 5 and 15 years and ESKD in one phenotypically girl with sex reversal developed at the age of 8 years. Conclusion We found that children with WT1-associated SRNS had a wide spectrum of renal and extrarenal phenotypes concerning the development of urogenital system and Wilms tumor with high risk of progression to CKD during childhood. Their phenotypes are clearly associated with the type and location of WT1 mutations led to DDS or FS. Genetic WT1 diagnostics is important in all children with SRNS for early detection, optimal treatment, tumor prevention and prognosis.

WT1 influences apoptosis and proliferation of immature mice granular cells through regulation of the wnt/β-catenin signal pathway

It was to study the influence of Wilms tumor suppressor gene (WT1) on ovarian granular cells (GCs) in mice, and the molecular mechanism involved. LV-WT1 short hairpin ribonucleic acid (shRNA) vector was used to downregulate WT1 expression in granular cells (GCs). The effects of WTI on proliferation and apoptosis of GCs were investigated. Western blot and qRT-PCR were used to assay the mRNA and protein expressions of Bax/bcl-2 in GCs transfected with LV-WT1-RNAi. The expression levels of SUZ12, Wnt5a, Wnt11, Wnt4, Wnt3a, Wnt2 mRNA in GCs were also determined. LV-WT1-RNAi significantly reduced WT1 expression, increased apoptosis and inhibited proliferation of GCs. The inhibition of WT1 had no significant effect on the expression of bcl-2 in GCs. The expressions of Wnt2, Wnt4 and Wnt5a were augmented in WT1-knockdown GCs, relative to non-transfected cells. WT1 activation is necessary for maintaining early survival of GCs in follicles via activation of the Wnt/β-catenin signal pathway.

Mitigation of premature ovarian failure by over-expression of lentivirus vector-mediated Wilms tumor-suppressor gene

Purpose: To investigate the effect of WT1(Wilms tumor-suppressor gene) overexpression on premature ovarian failure (POF)-mediated ovarian dysfunction. Methods: Three mice groups were used: control group (untreated mice), POF group (mice sterilized by intravenous injection of cyclophosphamide and busulfan), and POF-LV(lentiviral vector)􀀀GFP(green fluorescent protein)􀀀WT1 group (POF mice given intra-ovarian microinjection of LV􀀀GFP􀀀WT1, a WT1􀀀overexpressing lentiviral vector, one week after sterilization). Real time-PCR was employed to analyze in vitro WT1 overexpression levels. Overall ovarian function was measured by hormonal assay, H & E staining, and immuno-histochemical techniques. Results: Overexpression of WT1 in mice models of POF alleviated ovarian granulosa cell (GC) damage, increased ovary weight, and significantly increased follicular number (p < 0.05). Radioimmunoassays revealed reduction in plasma estradiol (E2) and follicle-stimulating hormone (FSH, p < 0.05). However, results from immune-histochemical assays showed reduced Bax expression levels, and increased expression of Bcl-2 in WTI-overexpression mice, relative to POF mice. Conclusion: Overexpression of WT1 may stimulate repair of ovarian tissue while improving endocrine function by inhibiting ovarian cell apoptosis signaling pathway in POF mice. Keywords: Premature ovarian failure, Wilms tumor suppressor gene, Chemotherapy, Apoptosis, Estradiol

Neuron-specific inactivation of Wt1 alters locomotion in mice and changes interneuron composition in the spinal cord.

Locomotion is coordinated by neuronal circuits of the spinal cord. Recently, dI6 neurons were shown to participate in the control of locomotion. A subpopulation of dI6 neurons expresses the Wilms tumor suppressor gene Wt1. However, the function of Wt1 in these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed a slower walk with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-/hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0, and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and that loss of Wt1 is associated with alterations in locomotion.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

Protein Kinase A/CREB Signaling Prevents Adriamycin-Induced Podocyte Apoptosis via Upregulation of Mitochondrial Respiratory Chain Complexes.

Previous work showed that the activation of protein kinase A (PKA) signaling promoted mitochondrial fusion and prevented podocyte apoptosis. The cAMP response element binding protein (CREB) is the main downstream transcription factor of PKA signaling. Here we show that the PKA agonist 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate-cyclic AMP (pCPT-cAMP) prevented the production of adriamycin (ADR)-induced reactive oxygen species and apoptosis in podocytes, which were inhibited by CREB RNA interference (RNAi). The activation of PKA enhanced mitochondrial function and prevented the ADR-induced decrease of mitochondrial respiratory chain complex I subunits, NADH-ubiquinone oxidoreductase complex (ND) 1/3/4 genes, and protein expression. Inhibition of CREB expression alleviated pCPT-cAMP-induced ND3, but not the recovery of ND1/4 protein, in ADR-treated podocytes. In addition, CREB RNAi blocked the pCPT-cAMP-induced increase in ATP and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α). The chromatin immunoprecipitation assay showed enrichment of CREB on PGC1-α and ND3 promoters, suggesting that these promoters are CREB targets. In vivo, both an endogenous cAMP activator (isoproterenol) and pCPT-cAMP decreased the albumin/creatinine ratio in mice with ADR nephropathy, reduced glomerular oxidative stress, and retained Wilm's tumor suppressor gene 1 (WT-1)-positive cells in glomeruli. We conclude that the upregulation of mitochondrial respiratory chain proteins played a partial role in the protection of PKA/CREB signaling.

Novel Function of a Transcription Factor WT1 in Regulating Decidualization in Human Endometrial Stromal Cells and Its Molecular Mechanism.

The Wilms tumor suppressor gene (WT1) encodes an essential transcription factor regulating mammalian urogenital development. However, the function of WT1 in human endometrium is still unclear. The current study examined the involvement of WT1 in the regulation of IGF-binding protein-1 (IGFBP-1) and prolactin (PRL), which are specific markers of decidualization, in human endometrial stromal cells (ESCs) undergoing decidualization. ESCs isolated from proliferative-phase endometrium were incubated with cyclic adenosine monophosphate (cAMP) to induce decidualization. cAMP increased WT1 expression with the induction of IGFBP-1 and PRL. Knockdown of WT1 by small interfering RNA inhibited cAMP-induced expression of IGFBP-1 and PRL. cAMP also induced the recruitment of WT1 to the IGFBP-1 and PRL promoters. To investigate the mechanism by which WT1 is upregulated by cAMP, we focused on C/EBPβ, a gene that regulates the expression of many genes during decidualization. Knockdown of C/EBPβ decreased cAMP-increased WT1 expression. cAMP increased the recruitment of C/EBPβ to the WT1 enhancer that is located approximately 14,000 bp downstream from the transcription start site. To test the endogenous function of the WT1 enhancer region on WT1 expression, the endogenous WT1 enhancer region was deleted by CRISPR/Cas9 system in HEK293 cells. The increase of WT1 expression by cAMP was not observed in the enhancer-deleted clones. Chromatin immunoprecipitation assay revealed that this enhancer region has high levels of H3K27ac and H3K4me1, which are active enhancer marks. These results show the role of WT1 in regulating decidualization in human ESCs. C/EBPβ is an upstream gene that regulates WT1 expression by binding to the novel enhancer region.

Aortopathy in Bicuspid Aortic Valve and Marfan Syndrome is Characterized by a Lack of Activation Potential of the Epicardium in the Ascending Aorta

Patients with a bicuspid aortic valve (BAV) and Marfan syndrome (MFS) have increased susceptibility for development ofaortopathy. In the heart, epicardial cells expressing Wilms tumor suppressor protein (Wt1) are known to become activated after myocardial infarction. We hypothesize that epicardium covering the aorta might show a similar response in BAV and MFS in pathologic conditions.Non- and dilated ascending aorta specimen of BAV (n = 36), tricuspid aortic valve (TAV) (n = 23), and MFS (8) were investigated. The aorta was studied by immunohistochemistry and immunofluorescence, using Wt1 and endothelial nitric oxide, which regulates Wt1 expression. Functionality and therefore activity of Wt1 was confirmed by co-expression with retinaldehyde dehydrogenase-II enzyme in the same cells.

Repression of CMIP transcription by WT1 is relevant to podocyte health

The WT1 (Wilm's tumor suppressor) gene is expressed throughout life in podocytes and is essential for the functional integrity of the glomerular filtration barrier. Wehave previously shown that CMIP (C-Maf inducing protein) is overproduced in podocyte diseases and alters intracellular signaling. Here we isolated the proximal region of the human CMIP promoter and showed by chromatin immunoprecipitation assays and electrophoretic-mobility shift that Wilm's tumor protein (WT1) bound to 2 WT1 response elements, located at positions -290/-274 and -57/-41 relative to transcription start site. Unlike the human CMIP gene, only one Wt1 response element was identified inthe mouse Cmip proximal promoter located at position -217/-206. Luciferase reporter assays indicated that WT1 dose-dependently inhibited the transcriptional induction of the CMIP promoter. Transfection of decoy oligonucleotides mimicking the WT1 response elements prevented the inhibition of WT1 on CMIP promoter activity. Furthermore, WT1 silencing promoted Cmip expression. In line with these findings, the abundance of Cmip was early and significantly increased at the transcript and protein level in podocytes displaying a primary defect in Wt1, including Denys-Drash syndrome and Frasier syndrome. Thus, WT1 is a major repressor of the CMIP gene in physiological situations, while conditional deletion of CMIP in the developing kidney did not affect the development of mature glomeruli.