Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.     

Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development.

To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3beta, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3beta seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCzeta mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCzeta-dependent regulation of the protein levels of p120 catenin and E-cadherin.     

Abnormal beta-catenin and reduced axin expression are associated with poor differentiation and progression in non-small cell lung cancer

We studied the expression of axin and beta-catenin and their relation to clinicopathologic factors in 100 non-small cell lung cancers (NSCLCs) by immunohistochemical analysis. The mutation in exon 3 of the beta-catenin gene was examined by polymerase chain reaction and direct sequencing. Preserved axin expression was significantly higher in well- and moderately differentiated NSCLC samples than in poorly differentiated ones. Reduced membranous expression of beta-catenin was shown in 80 cases, whereas 26 cases had aberrant nuclear expression. Poor differentiation and lymph node metastasis were associated significantly with reduced beta-catenin expression. Lower axin expression was related significantly to higher nuclear beta-catenin expression. However, this study failed to detect any exon 3 mutation in the beta-catenin gene in the 100 NSCLC samples. We conclude that reduced beta-catenin and axin expression might predict poor differentiation in NSCLC. Reduced axin expression, but not mutation in exon 3, might be an important explanation for the abnormal beta-catenin expression in NSCLC.     

The Par3/aPKC interaction is essential for end bud remodeling and progenitor differentiation during mammary gland morphogenesis

Mammalian polarity proteins have been studied predominantly in cell culture systems, and little is known about their functions in vivo. To address this issue, we used a shRNA lentiviral system to manipulate gene expression in mouse mammary stem/progenitor cells. Transplantation of Par3-depleted stem/progenitor cells into the mammary fat pad severely disrupted mammary development, and glands were characterized by ductal hyperplasia, luminal filling, and highly disorganized end bud structures that were unable to remodel into normal ductal structures. Unexpectedly, Par3-depleted mammary glands also had an expanded progenitor population. We identified a novel function for the atypical protein kinase C (aPKC)-binding domain of Par3 in restricting Par3 and aPKC to the apical region in mammary epithelia in vivo, and found that mammary morphogenesis is dependent on the ability of Par3 to directly bind aPKC. These results reveal a new function for Par3 in the regulation of progenitor differentiation and epithelial morphogenesis in vivo and demonstrate for the first time an essential requirement for the Par3–aPKC interaction.     

Tau overexpression in transgenic mice induces glycogen synthase kinase 3beta and beta-catenin phosphorylation

The abnormal phosphorylations of tau, GSK3beta, and beta-catenin have been shown to perform a crucial function in the neuropathology of Alzheimer's disease (AD). The primary objective of the current study was to determine the manner in which overexpressed htau23 interacts and regulates the behavior and phosphorylation characteristics of tau, GSK3beta, and beta-catenin. In order to accomplish this, transgenic mice expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE/htau23) were created. Transgenic mice evidenced the following: (i) tendency toward memory impairments at later stages, (ii) dramatic overexpression of the tau transgene, coupled with increased tau phosphorylation and paired helical filaments (PHFs), (iii) high levels of GSK3beta phosphorylation with advanced age, resulting in increases in the phosphorylations of tau and beta-catenin, (iv) an inhibitory effect of lithium on the phosphorylations of tau, GSK3beta, and beta-catenin, but not in the non-transgenic littermate group. Therefore, the overexpression of NSE/htau23 in the brains of transgenic mice induces abnormal phosphorylations of tau, GSK3beta, and beta-catenin, which are ultimately linked to neuronal degeneration in cases of AD. These transgenic mice are expected to prove useful for the development of new drugs for the treatment of AD.     

The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans

Axin, APC, and the kinase GSK3 beta are part of a destruction complex that regulates the stability of the Wnt pathway effector beta-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3 beta, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/beta-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway, pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/beta-catenin signaling in C. elegans.     

The Generation of Definitive Endoderm from Human Embryonic Stem Cells is Initially Independent from Activin A but Requires Canonical Wnt-Signaling

The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.     

Characterization of bipotential epidermal progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.     

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity

The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.     

Bone morphogenetic protein signaling inhibits hair follicle anagen induction by restricting epithelial stem/progenitor cell activation and expansion

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.     

Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells

BACKGROUND: Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. RESULTS: We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. CONCLUSION: These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.     

Functional link between retinoblastoma family of proteins and the Wnt signaling pathway in mouse epidermis.

The retinoblastoma family of proteins (pRb, p107, and p130) modulates cell cycle progression and differentiation of several tissues. We have demonstrated recently that p107 and p130 regulate keratinocyte terminal differentiation and hair follicle morphogenesis and development in vivo. This last aspect appears to be mediated by defective signaling from the mesenchyme and is associated with altered bone morphogenetic protein-4 (BMP4) -dependent signaling. However, many alterations were also found in the epithelial compartment. Given the importance of betacatenin in hair biology and in BMP signaling, we studied its expression in p107/p130-deficient skin. Although normal expression of betacatenin was found in p107/p130-deficient hair follicles, we found increased nuclear accumulation of betacatenin in the basal keratinocytes of the p107/p130-deficient mice skin. Biochemical analysis revealed that such an increase in betacatenin was due to the disruption of Axin/GSK3beta/betacatenin complexes promoted by the increased expression of Frat, the mouse homologue of GSK3betabinding protein (GBP), in epidermis, precluding the degradation of betacatenin. Collectively, these data represent the first evidence that retinoblastoma family and Wnt signaling pathways might be interconnected by functional links in skin.