Karin M. Kirschner

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene.

Neurotrophins and their cognate receptors play a pivotal role in the development and function of the nervous system. High expression levels of the neurotrophin receptor TrkB and its ligands in neuroblastomas are associated with an unfavorable outcome. We report here that NTRK2, which encodes the TrkB receptor tyrosine kinase, is an oxygen-regulated gene, whose expression is stimulated by the hypoxia-inducible factor-1 (HIF-1). TrkB mRNA and protein levels were elevated nearly 30-fold in neuroblastoma-derived Kelly cells in hypoxia (1% O2) versus normoxia (21% O2). A luciferase reporter construct containing ≈2.1 kilobases of the human TrkB promoter was activated about 6-fold both in hypoxia and after stimulation with the hypoxia mimetic 2,2′-dipyridyl (100 μm) at 21% O2. Luciferase activity in the presence of 2,2′-dipyridyl was reduced significantly upon small interfering RNA knockdown of HIF-1α but not of HIF-2α. Accordingly, hypoxia failed to stimulate the TrkB promoter in mouse embryonic fibroblasts that lacked HIF-1α. The hypoxia-responsive promoter region could be mapped to three HIF-1 binding elements that were located between -923 and -879 bp relative to the transcription start site. The migration of cultured neuroblastoma cells was increased ∼2-fold upon incubation at 1 versus 21% O2. This effect of hypoxia was abrogated with the tyrosine kinase inhibitor K252a (200 nm). Our findings indicate that transcription of the NTRK2 gene is stimulated at low oxygen tension through a HIF-1-dependent mechanism. In conclusion, enhanced expression of TrkB could represent a critical switch for the previously reported dedifferentiation of neuroblastoma cells under hypoxic conditions.

Translational Regulation of the Human Achaete-scute Homologue-1 by Fragile X Mental Retardation Protein

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5′-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)10 sequence located in the 5′-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.

The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the α4integrin Gene

Cell-matrix interaction through specific adhesion molecules is a critical step during organ development. In addition, down-regulation of cell adhesion receptors may promote tumor invasion and metastasis. We show here that the Wilms tumor suppressor Wt1, which is necessary for normal development of the epicardium, coronary vessels, genitourinary system, and other tissues, activates transcription of the α4integrin gene. Binding of the Wt1(-KTS) form, which is transcriptionally active, to the proximal α4integrin promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. A reporter construct harboring ∼1.9 kb of the human α4integrin gene promoter was activated significantly by transient co-transfection of a Wt1(-KTS) expression plasmid. Introducing mutations in two identified Wt1(-KTS) binding motifs in the proximal promoter of the α4integrin gene abrogated this stimulatory effect. Endogenous α4integrin transcripts were increased more than 3-fold in human embryonic kidney 293 cells with stable expression of the Wt1(-KTS) protein. Wt1-overexpressing cells showed augmented adhesion to the α4integrin ligand vascular cell adhesion molecule-1 that was abolished upon incubation with an inhibitory α4integrin antibody. Double immunofluorescent staining revealed co-localization of Wt1 and α4integrin in the developing epicardium of mouse embryos. Cardiac expression of α4integrin was reduced significantly in embryos with a homozygous Wt1 defect (Wt1-/-). These findings demonstrate that Wt1 can support cell adhesion through enhanced expression of α4integrin. This transcriptional activation of the α4integrin gene by Wt1(-KTS) might contribute to normal formation of the epicardium and other tissues in the developing embryo.

Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development

Background: ADAMTS16 is a mammalian metalloproteinase with unknown function. Results: Transcription of the Adamts16 gene is regulated by Wilms tumor protein Wt1, and knockdown of Adamts16 reduces branching morphogenesis in cultured embryonic kidneys. Conclusion: Adamts16 is a Wt1 target gene during murine genitourinary development. Significance: The findings provide novel insights into gene regulatory networks controlling kidney and gonad development. ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5′-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5′-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.

Oxygen-Dependent Gene Expression in Development and Cancer: Lessons Learned from the Wilms’ Tumor Gene, WT1

Adequate tissue oxygenation is a prerequisite for normal development of the embryo. Most fetal organs are exquisitely susceptible to hypoxia which occurs when the delivery of oxygen is exceeded by the actual demand. Developmental abnormalities due to insufficient supply with oxygen can result from the impaired expression of genes with essential functions during embryogenesis. As such, the Wilms’ tumor gene, WT1, is among the fetal genes that are regulated by the local oxygen tension. WT1 was originally discovered as a tumor suppressor gene owing to loss-of-function mutations in a subset of pediatric renal neoplasias, known as nephroblastomas or Wilms’ tumors. Wilms’ tumors can arise when pluripotent progenitor cells in the embryonic kidney continue to proliferate rather than differentiating to glomeruli and tubules. WT1 encodes a zinc finger protein, of which multiple isoforms exist due to alternative mRNA splicing in addition to translational and post-translational modifications. While some WT1 isoforms function as transcription factors, other WT1 proteins are presumably involved in post-transcriptional mRNA processing. However, the role of WT1 reaches far beyond that of a tumor suppressor as homozygous disruption of Wt1 in mice caused embryonic lethality with a failure of normal development of the kidneys, gonads, heart, and other tissues. WT1 mutations in humans are associated with malformation of the genitourinary system. A common paradigm of WT1 expressing cells is their capacity to switch between a mesenchymal and epithelial state. Thus, WT1 likely acts as a master switch that enables cells to undergo reciprocal epithelial-to-mesenchymal transition. Impairment of renal precursor cells to differentiate along the epithelial lineage due to WT1 mutations may favor malignant tumor growth. This article shall provide a concise review of the function of WT1 in development and disease with special consideration of its regulation by molecular oxygen.

Wilms’ tumor protein Wt1 regulates the Interleukin-10 (IL-10) gene

We identified the Wilms’ tumor protein, Wt1, as a novel transcriptional activator of the immunosuppressant cytokine interleukin‐10 (IL‐10). Silencing of Wt1 by RNA interference reduced IL‐10 mRNA levels by approximately 90%. IL‐10 transcripts were increased more than 15‐fold upon forced expression of Wt1. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a cis‐element that was responsible for activation of the IL‐10 promoter by Wt1 in murine macrophages. Mutation of the Wt1 binding motif abrogated stimulation of the IL‐10 promoter by tumor necrosis factor‐α (TNFα). These results suggest a novel immune regulatory function of Wt1 in controlling IL‐10 gene expression.

Nuclear Transport of Wilms′ Tumour Protein Wt1 Involves Importins α and β

Background/Aims: Wilms′ tumour protein, Wt1, is a zinc finger molecule, which is required for normal embryonic development. Mutations of the WT1 gene can give rise to childhood cancer of the kidneys. Different Wt1 isoforms exist, which function either as transcription factors or have a presumed role in mRNA processing. Previous studies suggested that Wt1 undergoes nucleocytoplasmic shuttling, and cytoplasmic Wt1 was higher in malignant than in normal cells. The aim of this study was to analyse the molecular pathways along which Wt1 shuttles between the cytoplasm and nucleus. Methods: Interaction of Wt1 protein with various importin α subtypes and importin β was assessed in pull-down assays and co-immunoprecipitation experiments. Nuclear localisation signals (NLS) were identified by combining site-directed mutagenesis with subcellular immunodetection of the transfected Wt1 variants. Results: Wt1(+/-KTS) proteins were found to interact with importin α1 and importin β in vitro and in living cells in vivo. A NLS that was necessary and sufficient for nuclear import could be mapped to the third Wt1 zinc finger. Mutation of this NLS strongly weakened binding of Wt1 to importins. Conclusion: Nuclear translocation of Wilms′ tumour protein involves importins α and β, and a NLS in the third zinc finger.

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.

Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation

Gonad morphogenesis relies on the correct spatiotemporal expression of a number of genes that together fulfill the differentiation of the bipotential gonad into testes or ovaries. As such, the transcription factors WT1 and GATA4 are pivotal for proper gonadal development. Here we address the contributions of GATA4 and WT1 to the sex differentiation phase in testes and ovaries. We applied an ex vivo technique for cultivating gonads in hanging droplets of media that were supplemented with vivo-morpholinos to knockdown WT1 and GATA4 either alone or in combination at the same developmental stage. We show that WT1 is equally important for both, the initial establishment and the maintenance of the sex-specific gene expression signature in testes and ovaries. We further identified Foxl2 as a novel putative downstream target gene of WT1. Moreover, knockdown of WT1 reduced mRNA levels of several molecular components of the hedgehog signaling pathway in XY gonads, whereas Gata4 vivo-morpholino treatment increased transcripts of Dhh and Ptch1 in embryonic testes. The data suggest that for its proper function, WT1 relies on the correct expression of the GATA4 protein. Furthermore, GATA4 down-regulates several ovarian promoting genes in testes, such as Ctnnb1, Fst, and Bmp2, suggesting that this repression is required for maintaining the male phenotype. In conclusion, this study provides novel insights into the role of WT1 and GATA4 during the sex differentiation phase and represents an approach that can be applied to assess other proteins with as yet unknown functions during gonadal development.

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.