Kyucheol Cho

Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix

The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited β-catenin-induced Topflash reporter activity and β-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, β-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of β-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/β-catenin activity. Our results add a layer of control to Wnt/β-catenin signaling in bone.

Transcription factor interactions and chromatin modifications associated with p53-mediated, developmental repression of the alpha-fetoprotein gene.

ABSTRACT We performed chromatin immunoprecipitation (ChIP) analyses of developmentally staged solid tissues isolated from wild-type and p53-null mice to determine specific histone N-terminal modifications, histone-modifying proteins, and transcription factor interactions at the developmental repressor region (−850) and core promoter of the hepatic tumor marker alpha-fetoprotein (AFP) gene. Both repression of AFP during liver development and silencing in the brain, where AFP is never expressed, are associated with dimethylation of histone H3 lysine 9 (DiMetH3K9) and the presence of heterochromatin protein 1 (HP1). These heterochromatic markers remain localized to AFP during developmental repression but spread to the upstream albumin gene during silencing. Developmentally regulated decreases in levels of acetylated H3 (AcH3K9) and H4 (AcH4) and of di- and trimethylated H3K4 (DiMetH3K4 and TriMetH3K4) occur at both the core promoter and distal repressor regions of AFP. Hepatic expression of AFP correlates with FoxA interaction at the repressor region and the binding of RNA polymerase II and TATA-binding protein to the core promoter. p53 acts as a developmental repressor of AFP in the liver by binding to chromatin, excluding FoxA interaction and targeting mSin3A/HDAC1 to the distal repressor region. p53-null mice exhibit developmentally delayed AFP repression, concomitant with acetylation of H3K9, methylation of H3K4, and loss of DiMetH3K9, mSin3A/HDAC1, and HP1 interactions.

Shared molecular mechanisms regulate multiple catenin proteins: canonical Wnt signals and components modulate p120-catenin isoform-1 and additional p120 subfamily members

Wnt signaling pathways have fundamental roles in animal development and tumor progression. Here, employing Xenopus embryos and mammalian cell lines, we report that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability through mechanisms shared with those regulating β-catenin. For example, in common with β-catenin, exogenous expression of destruction complex components, such as GSK3β and axin, promotes degradation of p120-catenin. Again in parallel with β-catenin, reduction of canonical Wnt signals upon depletion of LRP5 and LRP6 results in p120-catenin degradation. At the primary sequence level, we resolved conserved GSK3β phosphorylation sites in the amino-terminal region of p120-catenin present exclusively in isoform-1. Point-mutagenesis of these residues inhibited the association of destruction complex components, such as those involved in ubiquitylation, resulting in stabilization of p120-catenin. Functionally, in line with predictions, p120 stabilization increased its signaling activity in the context of the p120–Kaiso pathway. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and δ-catenin, associate with axin and are degraded in its presence. Thus, as supported using gain- and loss-of-function approaches in embryo and cell line systems, canonical Wnt signals appear poised to have an impact upon a breadth of catenin biology in vertebrate development and, possibly, human cancers.

Pronephric Tubulogenesis Requires Daam1-Mediated Planar Cell Polarity Signaling

Canonical β-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

Xenopus δ-catenin is essential in early embryogenesis and is functionally linked to cadherins and small GTPases

Catenins of the p120 subclass display an array of intracellular localizations and functions. Although the genetic knockout of mouse δ-catenin results in mild cognitive dysfunction, we found severe effects of its depletion in Xenopus. δ-catenin in Xenopus is transcribed as a full-length mRNA, or as three (or more) alternatively spliced isoforms designated A, B and C. Further structural and functional complexity is suggested by three predicted and alternative translation initiation sites. Transcript analysis suggests that each splice isoform is expressed during embryogenesis, with the B and C transcript levels varying according to developmental stage. Unlike the primarily neural expression of δ-catenin reported in mammals, δ-catenin is detectable in most adult Xenopus tissues, although it is enriched in neural structures. δ-catenin associates with classical cadherins, with crude embryo fractionations further revealing non-plasma-membrane pools that might be involved in cytoplasmic and/or nuclear functions. Depletion of δ-catenin caused gastrulation defects, phenotypes that were further enhanced by co-depletion of the related p120-catenin. Depletion was significantly rescued by titrated p120-catenin expression, suggesting that these catenins have shared roles. Biochemical assays indicated that δ-catenin depletion results in reduced cadherin levels and cell adhesion, as well as perturbation of RhoA and Rac1. Titrated doses of C-cadherin, dominant-negative RhoA or constitutively active Rac1 significantly rescued δ-catenin depletion. Collectively, our experiments indicate that δ-catenin has an essential role in amphibian development, and has functional links to cadherins and Rho-family GTPases.

Activation of Wnt Signaling by Chemically Induced Dimerization of LRP5 Disrupts Cellular Homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.

Xenopus Kazrin interacts with ARVCF-catenin, spectrin and p190B RhoGAP, and modulates RhoA activity and epithelial integrity

In common with other p120-catenin subfamily members, Xenopus ARVCF (xARVCF) binds cadherin cytoplasmic domains to enhance cadherin metabolic stability or, when dissociated, modulates Rho-family GTPases. We report here that xARVCF binds and is stabilized by Xenopus KazrinA (xKazrinA), a widely expressed conserved protein that bears little homology to established protein families, and which is known to influence keratinocyte proliferation and differentiation and cytoskeletal activity. Although we found that xKazrinA binds directly to xARVCF, we did not resolve xKazrinA within a larger ternary complex with cadherin, nor did it co-precipitate with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin, suggesting a potential means by which xKazrinA localizes to cell–cell borders. This was supported by the resolution of a ternary biochemical complex of xARVCF–xKazrinA–xβ2-spectrin and, in vivo, by the finding that ectodermal shedding followed depletion of xKazrin in Xenopus embryos, a phenotype partially rescued with exogenous xARVCF. Cell shedding appeared to be the consequence of RhoA activation, and thereby altered actin organization and cadherin function. Indeed, we also revealed that xKazrinA binds p190B RhoGAP, which was likewise capable of rescuing Kazrin depletion. Finally, xKazrinA was found to associate with δ-catenins and p0071-catenins but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin subfamily. Taken together, our study supports the essential role of Kazrin in development, and reveals the biochemical and functional association of KazrinA with ARVCF-catenin, spectrin and p190B RhoGAP.

Plakophilin-3 is required for late embryonic amphibian development, exhibiting roles in ectodermal and neural tissues.

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.

Kazrin, and its binding partners ARVCF‐ and delta‐catenin, are required for Xenopus laevis craniofacial development

The novel adaptor protein Kazrin associates with multifunctional entities including p120‐subfamily members (ARVCF‐, delta‐, and p0071‐catenin). Critical contributions of Kazrin to development or homeostasis are indicated with respect to ectoderm formation, integrity and keratinocyte differentiation, whereas its presence in varied tissues suggests broader roles. We find that Kazrin is maternally loaded, is expressed across development and becomes enriched in the forming head. Kazrins potential contributions to craniofacial development were probed by means of knockdown in the prospective anterior neural region. Cartilaginous head structures as well as eyes on injected sides were reduced in size, with molecular markers suggesting an impact upon neural crest cell establishment and migration. Similar effects followed the depletion of ARVCF (or delta‐catenin), with Kazrin:ARVCF functional interplay supported upon ARVCF partial rescue of Kazrin knockdown phenotypes. Thus, Kazrin and its associating ARVCF‐ and delta‐catenins, are required to form craniofacial tissues originating from cranial neural crest and precordal plate. Developmental Dynamics 240:2601–2612, 2011.