scholarly journals Alternative isoforms of KDM2A and KDM2B lysine demethylases negatively regulate canonical Wnt signaling

2020 ◽  
Author(s):  
Dijana Lađinović ◽  
Daniel Pinkas ◽  
Otakar Raška ◽  
František Liška ◽  
Ivan Raška ◽  
...  

AbstractA precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they are able to bind to activated promoters in order to repress them. We have observed that KDM2A-SF and KDM2B-SF bind to and repress the promoters of AXIN2 and CYCLIN D1, two canonical Wnt signaling target genes. Moreover, KDM2A-SF and KDM2B-SF can repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to affect the transcription of the TCF7L1 target genes also through this interaction.

2020 ◽  
Vol 295 (51) ◽  
pp. 17560-17572
Author(s):  
Siu Chiu Chan ◽  
Sachin S. Hajarnis ◽  
Sophia M. Vrba ◽  
Vishal Patel ◽  
Peter Igarashi

Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression of dominant-negative mutant HNF-1β in mIMCD3 cells produces hyperresponsiveness to exogenous Wnt ligands, which is inhibited by siRNA-mediated knockdown of Lef1. WT HNF-1β binds to two evolutionarily conserved sites located 94 and 30 kb from the mouse Lef1 promoter. Ablation of HNF-1β decreases H3K27 trimethylation repressive marks and increases β-catenin occupancy at a site 4 kb upstream to Lef1. Mechanistically, WT HNF-1β recruits the polycomb-repressive complex 2 that catalyzes H3K27 trimethylation. Deletion of the β-catenin–binding domain of LEF1 in HNF-1β–deficient cells abolishes the increase in Lef1 transcription and decreases the expression of downstream Wnt target genes. The canonical Wnt target gene, Axin2, is also a direct transcriptional target of HNF-1β through binding to negative regulatory elements in the gene promoter. These findings demonstrate that HNF-1β regulates canonical Wnt target genes through long-range effects on histone methylation at Wnt enhancers and reveal a new mode of active transcriptional repression by HNF-1β.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1851-1851
Author(s):  
Ya-Wei Qiang ◽  
Bo Hu ◽  
Yu Chen ◽  
Wei Qiang ◽  
Christoph Heuck ◽  
...  

Abstract Abstract 1851 Background: The proteasome inhibitor Bortezomib (Bz) shows significant activity in Multiple Myeloma (MM) by acting on MM cell directly as well as by augmenting bone formation in vitro and in vivo. Its effect on the bone could be traced to promoting differentiation of mesenchymal stem cells into osteoblast cells by regulating BMP2 and canonical Wnt signaling. However, the molecular mechanism mediating the direct anti-MM activity of Bz remains to be fully understood. Initially the rationale for the use of Bz in MM was inhibition of NF-kB signaling, yet subsequent studies showed that Bz actually induces activation of this pathway. In this study, we examined whether Bz regulates the activity of canonical Wnt signaling pathway in MM and whether the growth-inhibition effect of Bz was associated with activation of this pathway by using multiple MM cell lines including EJM, H929, INA6, KMS28BM, JJN3, L363, OPM1, OPM2, RPMI8226, UTMC, XG2 and XG6 as well as primary plasma cells (PC) from six patients with newly diagnosed MM. Methods/Results: Immunoblotting demonstrated that Bz induces stabilization of b-catenin protein in three MM cell lines (H929, OPM2 and UTMC) in a time- and dose-dependent manner. These changes were not seen when the same cell lysate were immunoblotted for other catenin family members, a-catenin and g-catenin. Increased levels of b-catenin protein response to Bz treatment were observed in other 9 MM cell lines (EJM, INA6, KMS28BM, JJN3, L363, OPM1, RPMI8226, XG2 and XG6) and in the 6 CD138+ sorted bone marrow PC from patients with MM. To determine if Bz regulation of b-catenin level is a specific effect of the inhibition of 26S proteasome subunit we treated the same MM cell lines with another proteasome inhibitor, MG132. Similar results were observed in response to MG132 for all four MM cell lines, suggesting the effect of Bz on b-catenin protein is 26S proteosome inhibitor specific. Increases in b-catenin protein levels in MM cells were not due to increased Ctnnb1/CTNNB (b-catenin) gene transcription as b-catenin mRNA did not change in these cells treated with Bz. These results indicate that proteasome inhibition increases b-catenin is independent of transcriptional upregulation. To determine whether Bz induces the nuclear localization and transcriptional activity of b-catenin, cells were incubated with Bz for 6 hours and then fractionated to separate the nuclear and cytoplasmic fractions. Treatment with Bz resulted an increase in nuclear b-catenin as well as b-catenin in cytoplasm in four cell lines including H929, INA6, OPM1 and MM144. Increase in cytoplasmic and nuclear b-catenin was further confirmed by immunofluorescence with antibodies specific for active form of b-catenin. To determine whether Bz affects b-catenin-mediated transcriptional activity, we used a TCF/LEF luciferase reporter construct cloned in lentiviral vector. OPM2 cells were infected with lentiviral particle containing the TCF reporter or containing empty vector and were then treated with serial concentrations of Bz. The luciferase activity exhibited a dose-dependent response to Bz analogous to the stabilization of b-catenin. Similar results were observed in 7 out of 8 MM cell lines compared with untreated control. Stimulation of TCF transcriptional activity by Bz was independent of modifiers of extracellular Wnt ligands, such as Frizzled receptors, LRP5/6 co-receptors and sFRPs or the activation of intracellular GSK3b. Conclusion: These results indicate that Bz augments activation of canonical Wnt signaling by preventing b-catenin protein from proteosome-mediated degradation in MM cells. Concentrations of Bz for stimulating TCF transcriptional activity are comparable to those being used to induce inhibition of MM proliferation. Experiments modulating cytoplasmic as well as the nuclear players and interactions of the Wnt-pathway are ongoing to determine if Bz mediated activation of b-catenin signaling is responsible for its direct anti-MM effect. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.


PLoS ONE ◽  
2020 ◽  
Vol 15 (10) ◽  
pp. e0236612
Author(s):  
Dijana Lađinović ◽  
Daniel Pinkas ◽  
Tijana Šopin ◽  
Otakar Raška ◽  
František Liška ◽  
...  

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 80
Author(s):  
Yentl Huybrechts ◽  
Eveline Boudin ◽  
Gretl Hendrickx ◽  
Ellen Steenackers ◽  
Neveen Hamdy ◽  
...  

Sclerosteosis is a high bone mass disorder, caused by pathogenic variants in the genes encoding sclerostin or LRP4. Both proteins form a complex that strongly inhibits canonical WNT signaling activity, a pathway of major importance in bone formation. So far, all reported disease-causing variants are located in the third β-propeller domain of LRP4, which is essential for the interaction with sclerostin. Here, we report the identification of two compound heterozygous variants, a known p.Arg1170Gln and a novel p.Arg632His variant, in a patient with a sclerosteosis phenotype. Interestingly, the novel variant is located in the first β-propeller domain, which is known to be indispensable for the interaction with agrin. However, using luciferase reporter assays, we demonstrated that both the p.Arg1170Gln and the p.Arg632His variant in LRP4 reduced the inhibitory capacity of sclerostin on canonical WNT signaling activity. In conclusion, this study is the first to demonstrate that a pathogenic variant in the first β-propeller domain of LRP4 can contribute to the development of sclerosteosis, which broadens the mutational spectrum of the disorder.


2020 ◽  
Author(s):  
Yueli Liu ◽  
Jiawen Wang ◽  
Corrigan Horton ◽  
Sol Katzman ◽  
Tao Cai ◽  
...  

AbstractBoth the canonical Wnt signaling and androgen signaling are important factors regulating prostate organogenesis. How these two pathways crosstalk to regulate prostate stem cell functions remain unclear. Here, we show that while canonical Wnt activity is required for prostate basal stem cell multipotency in vivo, ectopic Wnt activity does not promote basal-to-luminal cell differentiation. We provide evidence that androgen signaling may keep Wnt activity in check. In prostate organoid culture from basal cells, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated organoid growth in a concentration-dependent manner. Molecular analyses of organoids under different treatment conditions showed that androgen signaling down-regulated the expressions of a Wnt reporter as well as many Wnt target genes. Pathway analysis and gene set enrichment analysis of organoid RNA-seq data also revealed the canonical Wnt signaling as a key pathway distinguishing organoids treated with or without DHT. Notably, DHT treatment enhanced AR and β–catenin binding in the nuclei of prostate organoids, providing possible mechanistic clues. Our results reveal a critical role of AR signaling in modulating canonical Wnt activity in prostate basal cells to regulate their multipotency.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1193-1193
Author(s):  
Petr Danek ◽  
Miroslava Kardosova ◽  
Lucie Janeckova ◽  
Vladimir Korinek ◽  
Touati Benoukraf ◽  
...  

The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the Tcf/Lef transcription factors and subsequent transcriptional activation of Wnt-target genes. This pathway acts as an essential regulator of differentiation and cell fate decisions in various tissues. In the hematopoietic system, the function of the pathway has been investigated mainly by genetic manipulation of β-catenin. However, this approach does not allow to discriminate between Tcf/Lef dependent or independent β-catenin activity. In order to specifically identify the function of β-catenin-Tcf/Lef signaling in hematopoietic cells, we employed a transgenic mouse model expressing a dominant negative form of the human TCF4 transcription factor (dnTCF4). dnTCF4, a truncated protein lacking the β-catenin binding domain, abrogates activation of Wnt target genes, even when β-catenin is stabilized and translocated into the nucleus. In our model, expression of dnTCF4 is activated from the Rosa26 locus only in cells producing Cre recombinase (driven by Vav-iCre). Importantly, all components of Wnt signaling, including endogenous Tcf/Lef proteins and β-catenin, are intact in Cre-expressing cells. We observed that dnTCF4 transgenic mice have reduced numbers of granulocytes together with accumulation of short-term hematopoietic stem cells (ST-HSC) and common myeloid progenitors (CMPs) in bone marrow. Accordingly, dnTCF4-expressing bone marrow consistently showed a block of granulocytic differentiation and retention of an immature phenotype in colony forming assays. This differentiation arrest and accumulation of immature cells was also observed when wild type cells were cultured in semi-solid medium in the presence of a cell-penetrating peptide that disrupts β-catenin-Tcf/Lef interaction. Together, these results indicate that disruption of the β-catenin/Tcf-Lef interaction, either by genetic manipulation or a drug based approach, alters steady-state hematopoiesis. To identify a mechanism through which β-catenin-Tcf/Lef signaling affects granulopoiesis, wild type and dnTCF4 expressing ST-HSCs were subjected to RNA sequencing. Several genes related to myeloid development were differentially expressed in dnTCF4 expressing cells, including downregulation of Csf3r, the gene encoding for the G-CSF receptor. Publicly available datasets from ChIP-seq experiments on human cell lines confirmed TCF4 enrichment in the distal promoter of the CSF3R gene, suggesting that CSF3R is directly regulated by canonical Wnt signaling. Using flow cytometry we verified reduced levels of G-CSF receptor on the cell surface of dnTCF4 progenitor cells, and attenuation of downstream Stat3 phosphorylation after G-CSF treatment. Finally, when grown in the presence of G-CSF, dnTCF4-expressing bone marrow cells showed impaired differentiation abilities and reduced granulocytic counts compared to wild type bone marrow cells. These results encouraged us to investigate the role of the β-catenin-Tcf/Lef signaling pathway during emergency granulopoiesis by challenging mice with lipopolysaccharide (LPS). Remarkably, dnTCF4 mice showed defects upon LPS stimulation, and completely failed to maintain and expand myeloid progenitor populations, a critical step during emergency granulopoiesis. Altogether, we showed that β-catenin-Tcf/Lef signaling axis is crucial for proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Mechanistically, we demonstrated that the β-catenin-Tcf/Lef interaction controls expression of genes involved in myeloid differentiation, and directly enhances expression of the G-CSF receptor, a crucial molecule for proper development of granulocytes. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 556-556 ◽  
Author(s):  
Maulin Mukeshchandra Patel ◽  
Robert Silasi-Mansat ◽  
Ravi Shankar Keshari ◽  
Christopher L. Sansam ◽  
David A. Jones ◽  
...  

Abstract We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene's expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (>500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.


2020 ◽  
Vol 32 (3) ◽  
pp. 135-144
Author(s):  
Alfredo B. Cuellar-Barboza ◽  
Jorge A. Sánchez-Ruiz ◽  
Iram P. Rodriguez-Sanchez ◽  
Sarai González ◽  
Geovana Calvo ◽  
...  

AbstractBackground:Peripheral gene expression of several molecular pathways has been studied in major depressive disorder (MDD) with promising results. We sought to investigate some of these genes in a treatment-free Latino sample of Mexican descent.Material and Methods:The sample consisted of 50 MDD treatment-free cases and 50 sex and age-matched controls. Gene expression of candidate genes of neuroplasticity (BDNF, p11, and VGF), inflammation (IL1A, IL1B, IL4, IL6, IL7, IL8, IL10, MIF, and TNFA), the canonical Wnt signaling pathway (TCF7L2, APC, and GSK3B), and mTOR, was compared in cases and controls. RNA was obtained from blood samples. We used bivariate analyses to compare subjects versus control mean mRNA quantification of target genes and lineal regression modelling to test for effects of age and body mass index on gene expression.Results:Most subjects were female (66%) with a mean age of 26.7 (SD 7.9) years. Only GSK3B was differentially expressed between cases and controls at a statistically significant level (p = 0.048). TCF7L-2 showed the highest number of correlations with MDD-related traits, yet these were modest in size.Discussion:GSK3B encodes a moderator of the canonical Wnt signaling pathway. It has a role in neuroplasticity, neuroprotection, depression, and other psychiatric phenotypes. We found that adding population diversity has the potential to elicit distinct peripheral gene expression markers in MDD and MDD-related traits. However, our results should only be considered as hypothesis-generating research that merits further replication in larger cohorts of similar ancestry.


2011 ◽  
Vol 89 (6) ◽  
pp. 515-521 ◽  
Author(s):  
L. Li ◽  
L. Tam ◽  
L. Liu ◽  
T. Jin ◽  
D.S. Ng

Lysophosphatidic acid (LPA) is a bioactive phospholipid with a diverse range of biological activities including the modulation of adipogenesis. Treatment of 3T3-L1 cells and 3T3F44A cells with LPA inhibits adipogenesis and reduces expression of PPARγ through activation of RhoGTPase and its downstream Rho associated kinase (ROCK). The mechanism of suppression of PPARγ expression by Rho/ROCK is poorly understood. By treating the differentiating 3T3-L1 cells with various combinations of LPA and ROCK inhibitors, Y-27632 and fasudil, we observed that LPA treatment resulted in attenuation of adipogenesis and a significant reduction in PPARγ mRNA as early as 3 d post-induction. LPA treatment also resulted in significant but delayed upregulation of components of the canonical Wnt signaling, namely Wnt10b mRNA, β-catenin protein, and mRNA expression of β-catenin target genes, detectable at day 7, but not day 3. Treatment of the 3T3-L1 cells with ROCK inhibitors Y-27632 and fasudil revealed a tonic activation of β-catenin/target genes by ROCK. This study identified the existence of a novel cross talk between the Rho/ROCK pathway and the Wnt-signaling pathway. The LPA/Rho/ROCK pathway inhibits expression of PPARγ and adipogenesis in part through a delayed activation of the canonical Wnt-signaling pathway based on increased Wnt10b expression and β-catenin induction.


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