miRNA let-7e Modulates the Wnt Pathway and Early Nephrogenic Markers in Mouse Embryonic Stem Cell Differentiation

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3β^P) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3β^P and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.     

Adenomatous Polyposis Coli Tumor Suppressor Protein Has Signaling Activity in Xenopus laevis Embryos Resulting in the Induction of an Ectopic Dorsoanterior Axis

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein β-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic β-catenin expression. In fact, axis induction by APC required the availability of cytosolic β-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of β-catenin, indicating that it has direct positive signaling activity in addition to its role in β-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/β-catenin signaling pathway, either upstream of, or in conjunction with, β-catenin.     

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference.     

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation,Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4^−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc ^Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.     

WNT4/β-Catenin Pathway Maintains Female Germ Cell Survival by Inhibiting Activin βB in the Mouse Fetal Ovary

Female germ cells are essential for organogenesis of the ovary; without them, ovarian follicles do not form and functional and structural characteristics of the ovary are lost. We and others showed previously that when either Wnt4 or β-catenin was inactivated in the fetal ovary, female germ cells underwent degeneration. In this study, we set out to understand whether these two factors belong to the same pathway and how they maintain female germ cell survival. We found that activation of β-catenin in somatic cells in the Wnt4 knockout ovary restored germ cell numbers, placing β-catenin downstream of WNT4. In the absence of Wnt4 or β-catenin, female germ cells entered meiosis properly; however, they underwent apoptosis afterwards. Activin βB (Inhbb), a subunit of activins, was upregulated in the Wnt4 and β-catenin knockout ovaries, suggesting that Inhbb could be the cause for the loss of female germ cells, which are positive for activin receptors. Indeed, removal of Inhbb in the Wnt4 knockout ovaries prevented female germ cells from undergoing degeneration. We conclude that WNT4 maintains female germ cell survival by inhibiting Inhbb expression via β-catenin in the somatic cells. Maintenance of female germ cells hinge upon a delicate balance between positive (WNT4 and β-catenin) and negative (activin βB) regulators derived from the somatic cells in the fetal ovary.     

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.