The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6.

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/ß-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

The USP46 complex deubiquitylates LRP6 to promote Wnt/ß-catenin signaling.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

USP47 deubiquitylates Groucho/TLE to promote Wnt-ß-catenin signaling.

The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization.

Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. Herein, we tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. We find that the critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease.

Developmental regulation of Wnt signaling by Nagk and the UDP-GlcNAc salvage pathway.

In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFß, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.

Wingless/Wnt Signaling in Intestinal Development, Homeostasis, Regeneration and Tumorigenesis: A Drosophila Perspective.

In mammals, the Wnt/ß-catenin signal transduction pathway regulates intestinal stem cell maintenance and proliferation, whereas Wnt pathway hyperactivation, resulting primarily from the inactivation of the tumor suppressor Adenomatous polyposis coli (APC), triggers the development of the vast majority of colorectal cancers. The Drosophila adult gut has recently emerged as a powerful model to elucidate the mechanisms by which Wingless/Wnt signaling regulates intestinal development, homeostasis, regeneration, and tumorigenesis. Herein, we review recent insights on the roles of Wnt signaling in Drosophila intestinal physiology and pathology.

APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway.

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting ß-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased ß-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote ß-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC.

The aberrant activation of Wnt signal transduction initiates the development of 90% of colorectal cancers, the majority of which arise from inactivation of the tumor suppressor Adenomatous polyposis coli (APC). In the classical model for Wnt signaling, the primary role of APC is to act, together with the concentration-limiting scaffold protein Axin, in a "destruction complex" that directs the phosphorylation and consequent proteasomal degradation of the transcriptional activator ß-catenin, thereby preventing signaling in the Wnt-off state. Following Wnt stimulation, Axin is recruited to a multiprotein "signalosome" required for pathway activation. Whereas it is well-documented that APC is essential in the destruction complex, APC's role in this complex remains elusive. Here, we demonstrate in Drosophila that Axin exists in two distinct phosphorylation states in Wnt-off and Wnt-on conditions, respectively, that underlie its roles in the destruction complex and signalosome. These two Axin phosphorylation states are catalyzed by glycogen synthase kinase 3 (GSK3), and unexpectedly, completely dependent on APC in both unstimulated and Wnt-stimulated conditions. In a major revision of the classical model, we show that APC is essential not only in the destruction complex, but also for the rapid transition in Axin that occurs after Wnt stimulation and Axin's subsequent association with the Wnt co-receptor LRP6/Arrow, one of the earliest steps in pathway activation. We propose that this novel requirement for APC in Axin regulation through phosphorylation both prevents signaling in the Wnt-off state and promotes signaling immediately following Wnt stimulation.

Intestinal stem cell overproliferation resulting from inactivation of the APC tumor suppressor requires the transcription cofactors Earthbound and Erect wing.

Wnt/ß-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). The ß-catenin-TCF transcription complex activates both the physiological expression of Wnt target genes in the normal intestinal epithelium and their aberrantly increased expression in colorectal tumors. Whether mechanistic differences in the Wnt transcription machinery drive these distinct levels of target gene activation in physiological versus pathological states remains uncertain, but is relevant for the design of new therapeutic strategies. Here, using a Drosophila model, we demonstrate that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers. Together, our findings reveal distinct requirements for Ebd and Ewg in physiological Wnt pathway activation versus oncogenic Wnt pathway hyperactivation following Apc1 loss. Such differentially utilized transcription cofactors may offer new opportunities for the selective targeting of Wnt-driven cancers.

Wnt pathway activation by ADP-ribosylation.

Wnt/ß-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly.

The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila.

Wnt/ß-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer.

Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries.

Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/ß-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, we demonstrate that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. We conclude that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries.

Regulation of TGFß receptor trafficking and signaling by atypical protein kinase C.

Transforming growth factor beta (TGFß) signaling is linked to the membrane trafficking of TGFß receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFß receptors, and further explored which PKC isoforms may be responsible for altered TGFß signaling patterns. Using immunofluorescence microscopy and (125)I-TGFß internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFß receptor trafficking and delays TGFß receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFß-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFß receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFß receptor degradation and extend TGFß signaling. Our findings suggest that atypical PKC isoforms regulate TGFß signaling by altering cell surface TGFß receptor trafficking and degradation.

Erect Wing facilitates context-dependent Wnt/Wingless signaling by recruiting the cell-specific Armadillo-TCF adaptor Earthbound to chromatin.

During metazoan development, the Wnt/Wingless signal transduction pathway is activated repetitively to direct cell proliferation, fate specification, differentiation and apoptosis. Distinct outcomes are elicited by Wnt stimulation in different cellular contexts; however, mechanisms that confer context specificity to Wnt signaling responses remain largely unknown. Starting with an unbiased forward genetic screen in Drosophila, we recently uncovered a novel mechanism by which the cell-specific co-factor Earthbound 1 (Ebd1), and its human homolog jerky, promote interaction between the Wnt pathway transcriptional co-activators ß-catenin/Armadillo and TCF to facilitate context-dependent Wnt signaling responses. Here, through the same genetic screen, we find an unanticipated requirement for Erect Wing (Ewg), the fly homolog of the human sequence-specific DNA-binding transcriptional activator nuclear respiratory factor 1 (NRF1), in promoting contextual regulation of Wingless signaling. Ewg and Ebd1 functionally interact with the Armadillo-TCF complex and mediate the same context-dependent Wingless signaling responses. In addition, Ewg and Ebd1 have similar cell-specific expression profiles, bind to each other directly and also associate with chromatin at shared genomic sites. Furthermore, recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. Our findings provide in vivo evidence that recruitment of a cell-specific co-factor complex to specific chromatin sites, coupled with its ability to facilitate Armadillo-TCF interaction and transcriptional activity, promotes contextual regulation of Wnt/Wingless signaling responses.

Jerky/Earthbound facilitates cell-specific Wnt/Wingless signalling by modulating ß-catenin-TCF activity.

Wnt/Wingless signal transduction directs fundamental developmental processes, and upon hyperactivation triggers colorectal adenoma/carcinoma formation. Responses to Wnt stimulation are cell specific and diverse; yet, how cell context modulates Wnt signalling outcome remains obscure. In a Drosophila genetic screen for components that promote Wingless signalling, we identified Earthbound 1 (Ebd1), a novel member in a protein family containing Centromere Binding Protein B (CENPB)-type DNA binding domains. Ebd1 is expressed in only a subset of Wingless responsive cell types, and is required for only a limited number of Wingless-dependent processes. In addition, Ebd1 shares sequence similarity and can be functionally replaced with the human CENPB domain protein Jerky, previously implicated in juvenile myoclonic epilepsy development. Both Jerky and Ebd1 interact directly with the Wnt/Wingless pathway transcriptional co-activators ß-catenin/Armadillo and T-cell factor (TCF). In colon carcinoma cells, Jerky facilitates Wnt signalling by promoting association of ß-catenin with TCF and recruitment of ß-catenin to chromatin. These findings indicate that tissue-restricted transcriptional co-activators facilitate cell-specific Wnt/Wingless signalling responses by modulating ß-catenin-TCF activity.

The adenomatous polyposis coli tumor suppressor and Wnt signaling in the regulation of apoptosis.

The adenomatous polyposis coli (APC) tumor suppressor is an essential negative regulator in the evolutionarily conserved Wnt/Wingless (Wg) signal transduction pathway. During normal development, Wnt signaling is required not only to induce cell proliferation and cell fate specification, but also to induce apoptotic cell death. However in some malignant states triggered by APC loss, inappropriate activation of Wnt signaling promotes cell survival and inhibits cell death, indicating that the cellular response to APC loss and Wnt signaling is highly dependent on cell context. This chapter summarizes our current understanding of the role of APC and Wnt signaling in the regulation of apoptosis, based upon studies from fly and mouse in vivo models, as well as cultured carcinoma cells.

Adenomatous polyposis coli is present near the minimal level required for accurate graded responses to the Wingless morphogen.

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, we reduced Apc activity to different degrees, and analyzed the effects on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. We find that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. We conclude that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen.

Dual positive and negative regulation of wingless signaling by adenomatous polyposis coli.

The evolutionarily conserved Wnt/Wingless signal transduction pathway directs cell proliferation, cell fate, and cell death during development in metazoans and is inappropriately activated in several types of cancer. The majority of colorectal carcinomas contain truncating mutations in the adenomatous polyposis coli (APC) tumor suppressor, a negative regulator of Wnt/Wingless signaling. Here, we demonstrate that Drosophila Apc homologs also have an activating role in both physiological and ectopic Wingless signaling. The Apc amino terminus is important for its activating function, whereas the beta-catenin binding sites are dispensable. Apc likely promotes Wingless transduction through down-regulation of Axin, a negative regulator of Wingless signaling. Given the evolutionary conservation of APC in Wnt signal transduction, an activating role may also be present in vertebrates with relevance to development and cancer.

GATA- and Smad1-dependent enhancers in the Smad7 gene differentially interpret bone morphogenetic protein concentrations.

Smad7, an inhibitor of transforming growth factor beta superfamily signaling, is induced by bone morphogenetic protein (BMP) in an inhibitory feedback loop. Here, we identify multiple BMP response elements (BREs) in the Smad7 gene and demonstrate that they function differentially to interpret BMP signals in a cell type-specific manner. Two BREs (BRE-1 and -2) reside in the promoter region. One of these contains several conserved Smad1 and Smad4 binding sites that cooperate to mediate BMP-dependent induction, most likely in the absence of DNA binding partners. The third BRE (I-BRE) resides in the first intron and contains GATA factor binding sites. GATA-1, -5, or -6 is required for strong activation of I-BRE, and we show that they assemble with Smad1 on the I-BRE in living cells. Activation of the I-BRE is mediated by a specific region in GATA-5 and -6 but does not require direct physical interaction with Smad1. Comparison of I-BRE to BRE-1 showed that I-BRE is more responsive to low BMP concentrations. Moreover, analysis by chromatin immunoprecipitation experiments demonstrates that the endogenous I-BRE is occupied more robustly by endogenous Smad1 than is BRE-1. This correlates with regulation of the Smad7 gene, which is induced at lower BMP concentrations in GATA-expressing cell lines compared to non-GATA-expressing lines. These data thus define how cooperative and noncooperative Smad-dependent transcriptional regulation can function to interpret different BMP concentrations.