Ken-Ichi Takemaru

Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity

β-Catenin functions in both cell–cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of β-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although β-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits β-catenin–mediated transcriptional activation. Here, we identified 14-3-3ε and 14-3-3ζ as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3–binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with β-catenin, causing β-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of β-catenin, thereby antagonizing β-catenin signaling.

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

A role for primary cilia in glutamatergic synaptic integration of adult-born neurons

The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule-based signaling center, is essential for vertebrate development, including the development of the CNS. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born mouse hippocampal neurons. Primary cilia were absent in young adult-born neurons, but assembled precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional deletion of cilia from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Deletion of primary cilia led to enhanced Wnt and β-catenin signaling, which may account for these developmental defects. Taken together, our findings identify the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.

Chibby, an Antagonist of the Wnt/β-Catenin Pathway, Facilitates Cardiomyocyte Differentiation of Murine Embryonic Stem Cells

Background— Embryonic stem cell (ESC)–derived cardiomyocytes are anticipated to serve as a useful source for future cell-based cardiovascular disease therapies. Research emphasis is currently focused on determining methods to direct the differentiation of ESCs to a large population of cardiomyocytes with high purity. To this aim, understanding the molecular mechanisms that control ESC-to-cardiomyocyte differentiation should play a critical role in the development of this methodology. The Wnt/β-catenin signaling pathway has been implicated in both embryonic cardiac development and in vitro ESC differentiation into cardiomyocytes. Chibby is a recently identified nuclear protein that directly binds to β-catenin and antagonizes its transcriptional activity. Methods and Results— Chibby was ubiquitously expressed in early stages of ESC differentiation but upregulated during cardiomyocyte specification. Of interest, the Chibby gene promoter has multiple binding sites for the cardiac-specific homeodomain protein Nkx2.5, and its promoter activity was indeed positively regulated by Nkx2.5. Furthermore, overexpression of Chibby increased cardiac differentiation of ESCs, whereas loss of Chibby by RNAi impaired cardiomyocyte differentiation. Conclusions— These data illustrate the regulation and function of Chibby in facilitating cardiomyocyte differentiation from ESCs. By revealing molecular mechanisms that control ESC-to-cardiomyocyte differentiation, this study will allow for the future development of technologies to improve cardiomyocyte differentiation from ESCs.

An Oncogenic Hub: β-Catenin as a Molecular Target for Cancer Therapeutics

The Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and in maintenance of organs and tissues in adults. Activation of this signaling cascade inhibits degradation of the pivotal component beta-catenin, which in turn stimulates transcription of downstream target genes. Over the past two decades, intensive worldwide investigations have yielded considerable progress toward understanding the cellular and molecular mechanisms of Wnt signaling and its involvement in the pathogenesis of a range of human diseases. Remarkably, beta-catenin signaling is aberrantly activated in greater than 70% of colorectal cancers and to a lesser extent in other tumor types, promoting cancer cell proliferation, survival and migration. Accordingly, beta-catenin has gained recognition as an enticing molecular target for cancer therapeutics. Disruption of protein-protein interactions essential for beta-catenin activity holds immense promise for the development of novel anti-cancer drugs. In this review, we focus on the regulation of beta-catenin-dependent transcriptional activation and discuss potential therapeutic opportunities to block this signaling pathway in cancer.

Nuclear-Cytoplasmic Shuttling of Chibby Controls β-Catenin Signaling

Chibby (Cby) acts with 14-3-3 to regulate β-catenin localization in the canonical Wnt pathway. We show that Cby harbors functional NLS and NES motifs, and shuttles between the nucleus and cytoplasm. Cby distribution at steady state is controlled by an intricate cooperation between 14-3-3, CRM1 and importin-α, which impacts on β-catenin signaling.

Abrogation of β-catenin signaling in oligodendrocyte precursor cells reduces glial scarring and promotes axon regeneration after CNS injury.

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted β-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing β-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration.

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

In the early stages of cilia formation in the mouse airway epithelium, Chibby is recruited to the distal appendages of centrioles and is necessary for efficient ciliary vesicle formation and basal body docking at the apical cell membrane.

Fine-tuning of nuclear β-catenin by Chibby and 14-3-3

Chibby (Cby) is an evolutionarily conserved antagonist of β -catenin, a central player of the canonical Wnt signaling pathway, which acts as a transcriptional coactivator. Cby physically interacts with the C-terminal activation domain of β -catenin and blocks its transcriptional activation potential through competition with DNA-binding Tcf/Lef transcription factors. Our recent study revealed a second mechanism for Cby-mediated β -catenin inhibition in which Cby cooperates with 14-3-3 adaptor proteins to facilitate nuclear export of β -catenin, following phosphorylation of Cby by Akt kinase. Therefore, our findings unravel a novel molecular mechanism regulating the dynamic nucleo-cytoplamic trafficking of β -catenin and provide new insights into the cross-talk between the Wnt and Akt signaling pathways. Here, we review recent literature concerning Cby function and discuss our current understanding of the relationship between Wnt and Akt signaling.

Altered Lung Morphogenesis, Epithelial Cell Differentiation and Mechanics in Mice Deficient in the Wnt/β-Catenin Antagonist Chibby

The canonical Wnt/β-catenin pathway plays crucial roles in various aspects of lung morphogenesis and regeneration/repair. Here, we examined the lung phenotype and function in mice lacking the Wnt/β-catenin antagonist Chibby (Cby). In support of its inhibitory role in canonical Wnt signaling, expression of β-catenin target genes is elevated in the Cby−/− lung. Notably, Cby protein is prominently associated with the centrosome/basal body microtubule structures in embryonic lung epithelial progenitor cells, and later enriches as discrete foci at the base of motile cilia in airway ciliated cells. At birth, Cby−/− lungs are grossly normal but spontaneously develop alveolar airspace enlargement with reduced proliferation and abnormal differentiation of lung epithelial cells, resulting in altered pulmonary function. Consistent with the Cby expression pattern, airway ciliated cells exhibit a marked paucity of motile cilia with apparent failure of basal body docking. Moreover, we demonstrate that Cby is a direct downstream target for the master ciliogenesis transcription factor Foxj1. Collectively, our results demonstrate that Cby facilitates proper postnatal lung development and function.