Tomas Valenta

The many faces and functions of β-catenin

β‐Catenin (Armadillo in Drosophila) is a multitasking and evolutionary conserved molecule that in metazoans exerts a crucial role in a multitude of developmental and homeostatic processes. More specifically, β‐catenin is an integral structural component of cadherin‐based adherens junctions, and the key nuclear effector of canonical Wnt signalling in the nucleus. Imbalance in the structural and signalling properties of β‐catenin often results in disease and deregulated growth connected to cancer and metastasis. Intense research into the life of β‐catenin has revealed a complex picture. Here, we try to capture the state of the art: we try to summarize and make some sense of the processes that regulate β‐catenin, as well as the plethora of β‐catenin binding partners. One focus will be the interaction of β‐catenin with different transcription factors and the potential implications of these interactions for direct cross‐talk between β‐catenin and non‐Wnt signalling pathways.

HIC1 attenuates Wnt signaling by recruitment of TCF-4 and β-catenin to the nuclear bodies

The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller–Dieker syndrome. HIC1 encodes a zinc‐finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N‐terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/β‐catenin‐mediated transcription in Wnt‐stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF‐4 and to recruit TCF‐4 and β‐catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF‐binding elements of the Wnt‐responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/β‐catenin signaling.

Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling.

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

Probing transcription-specific outputs of β-catenin in vivo

β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/β-catenin signaling in dorsal neural tube development. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity.

Canonical Wnt Signaling Regulates Atrioventricular Junction Programming and Electrophysiological Properties

Rationale: Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. Objective: To determine the role of canonical Wnt signaling in the myocardium during AVC development. Methods and Results: We used a novel allele of &bgr;-catenin that preserves &bgr;-catenin’s cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff–Parkinson–White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of &bgr;-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression. Conclusions: Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue.

Transforming growth factor-β-dependent Wnt secretion controls myofibroblast formation and myocardial fibrosis progression in experimental autoimmune myocarditis

Aims Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-&bgr; (TGF-&bgr;) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. Methods and results Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/&bgr;-catenin pathway in response to TGF-&bgr; signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-&bgr;-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/&bgr;-catenin-mediated transcription with ICG-001 or genetic loss of &bgr;-catenin also prevented TGF-&bgr;-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-&bgr;-activated kinase 1 (TAK1) pathway completely abrogated TGF-&bgr;-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-&bgr;, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/&bgr;-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. Conclusion We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-&bgr;-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.

DIFFERING SENSITIVITY OF TUMOR CELLS TO APOPTOSIS INDUCED BY IRON DEPRIVATION IN VITRO

SummaryWe studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 μM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EL4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.

Wnt/β‐Catenin Signaling Regulates Sequential Fate Decisions of Murine Cortical Precursor Cells

The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. β‐Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of β‐catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel β‐catenin mutant allele with conditional inactivation approaches. Wnt/β‐catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/β‐catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper‐layer neurons and later, to precocious astrogenesis. Indeed, a genome‐wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/β‐catenin signaling‐ablated cortex. Thus, β‐catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage‐specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain. Stem Cells 2015;33:170–182

Dazap2 modulates transcription driven by the Wnt effector TCF-4

A major outcome of the canonical Wnt/β-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with β-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/β-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.

Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFκB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies.