- Email: [email protected]
LRP6 Signaling
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Review
b-Catenin-Independent Roles of Wnt/LRP6 Signaling Sergio P. Acebron1,* and Christof Niehrs1,2,* Wnt/LRP6 signaling is best known for the b-catenin-dependent regulation of target genes. However, pathway branches have recently emerged, including Wnt/STOP signaling, which act independently of b-catenin and transcription. We review here the molecular mechanisms underlying b-catenin-independent Wnt/LRP6 signaling cascades and their implications for cell biology, development, and physiology.
Wnt/LRP6 Signaling Wnt proteins are secreted short-range signals found in all metazoans, which function in embryonic development and adult tissue homeostasis by regulating fate, proliferation, polarity, and migration of cells [1–3]. Misregulation of Wnt signaling can lead to disease, notably cancer [4,5]. In most mammals there are 19 Wnt genes that can signal through a variety of receptors and downstream pathways (reviewed in [6]). Roughly, Wnt signaling is subdivided into a canonical pathway, also known as the Wnt/b-catenin cascade, and a non-canonical pathway, the most prominent of which is the Wnt/PCP (planar cell polarity) pathway (reviewed in [7]). Most cellular Wnt signaling is transmitted via seven transmembrane receptors of the Frizzled (Fzd) family [6]. A characteristic feature of Wnt/b-catenin signaling is that it not only involves Fzd receptors but additionally the Wnt coreceptors lipoprotein receptor-related proteins 5,6 (LRP5 and LRP6). Wnt binding to Fzd/LRP6 activates what has traditionally been viewed as a linear signal transduction cascade, which culminates in the stabilization of b-catenin. However, it has become clear that this pathway branches off to trigger a variety of other processes which are independent of b-catenin-mediated transcription (Figure 1A and Box 1). The term Wnt/LRP6 signaling encompasses all these downstream events, including but not limited to Wnt/b-catenin signaling, also known as canonical Wnt signaling. We review here the different b-catenin-independent events triggered by Wnt/LRP6 signaling and their role in cell biology, development, and physiology. Reviews focused on non-canonical Wnt signaling can be found elsewhere [3,8,9].
Trends Wnt/LRP6 signaling is thought to proceed by b-catenin mediated transcription. However, this pathway branches off to trigger a variety of other processes, revising the traditional transcription-centric view. Wnt/STOP signaling is one such novel branch of Wnt/LRP6 signaling, which peaks during mitosis to slow down protein degradation as cells prepare to divide. Transcription-independent Wnt/LRP6 signaling regulates cell growth and mitotic progression in dividing cells, axonal remodeling in post-mitotic neurons, and maturation of germ cells.
The Upstream Cascade Upon Wnt ligand binding, Fzd/LRP6 receptors cluster together with the scaffold protein Dishevelled (Dvl) into signalosomes, which are endocytosed [10–12] (Figure 1B). Signalosome formation induces LRP6 phosphorylation by glycogen synthase kinase 3 (GSK3) at PPPSP motifs, which primes adjacent S/T clusters for phosphorylation by casein kinase 1g (CK1g) [13,14]. In addition to GSK3, other kinases phosphorylate the LRP6 PPPSP motifs in a Wnt-independent manner (reviewed in [15]). For instance, the competence of LRP6 to respond to Wnt ligands is maximal during G2/M owing to primed phosphorylation by cyclin-dependent kinase 14 (CDK14/ PFTK1) and its regulators, mitotic cyclin Y and cyclin Y-like 1 (CCNY/CCNYL1) [16–19]. Phosphorylated LRP6 recruits a multiprotein ‘destruction complex’ that contains the scaffold proteins Axin and adenomatous polyposis coli (APC), the kinases CK1/ and GSK3, and the
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1 Division of Molecular Embryology, Deutsches Krebsforschungszentrum (DKFZ)–Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) Alliance, 69120 Heidelberg, Germany 2 Institute of Molecular Biology, 55128 Mainz, Germany
*Correspondence: [email protected] (S.P. Acebron) and [email protected] (C. Niehrs).
http://dx.doi.org/10.1016/j.tcb.2016.07.009 © 2016 Elsevier Ltd. All rights reserved.
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(A)
Wnts ‘Non-canonical’ Wnt/PCP, Wnt/Ca+2
Wnt/LRP6 signaling ‘Canonical’
β-catenin independent signaling
Wnt/β-catenin signaling
Wnt/STOP signaling
Other: e.g., Wnt/TOR
(B) Wnt
Fzd
LRP6 DvI Signalosome
MVB
β-catenin destrucon complex GSK3
Wnt/β-catenin signaling GSK3
Wnt/STOP signaling
GSK3 SK
GSK3 SK
20 S
β-Cat β-Cat
Wnt/TOR signaling
TSC2
P
20 S
TSC2 Protein stabilizaon
β-Cat
Ribosome β-Cat
Gene transcripon
TORC1
TCF
Protein translaon
Cell growth Cell division
Figure 1. Wnt Signaling Pathways. (A) Hierarchy of different Wnt signaling cascades. (B) Model of the Wnt/LRP6 signaling pathway. Wnt ligands form a ternary complex with LRP6 and Frizzled[3_TD$IF] (Fzd), which cluster on platforms of Dvl. These clusters are internalized in signalosomes, and recruit the destruction complex. LRP6 signalosomes further mature into multivesicular bodies where they sequester GSK3. Wnt/LRP6 signaling triggers several downstream cascades. In Wnt/ b-catenin signaling it leads to the stabilization of b-catenin (b-Cat), which upregulates the transcription of target genes. In Wnt/TOR signaling it blocks GSK3-dependent phosphorylation of TSC2, thereby derepressing TORC1 and promoting protein translation. In Wnt/STOP signaling it stabilizes GSK3 targets which are required for example for cell growth and division. Note that Wnt/LRP6 signaling can modulate the activity of other GSK3 targets in addition to TSC2 (Table 1[4_TD$IF]) Proteasome (20 S).
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Box 1. A Practical Guide to Branches of Wnt/LRP6 Schematic of experimental flow (1–5) to assess involvement of specific downstream cascades in a Wnt-induced response[9_TD$IF] (Figure I). Note that these assays serve only as a guideline for first analysis and that full assignment to a given cascade may require further ongoing investigations for the directness of effects. For individual examples see Table [10_TD$IF]1 in main text. (1) Wnt3a is well established in Wnt/LRP6 signaling and the recombinant protein is commercially available, or can be used as conditioned medium. Dkk1 inhibits Wnt/LRP6 signaling [95], and is commercially available. (2) 6Bromoindirubin-3’-oxime (BIO) is a chemical inhibitor of GSK3. (3) GSK3 targets that do not show increased protein levels upon Wnt3a or BIO may be directly activity-modulated. Phosphorylation status can be analyzed, for example by phosphatase- and BIO-sensitive mobility shift in 1D or 2D gels. (4) Dependency on b-catenin can be analyzed by overexpression or depletion of b-catenin, or by using the b-catenin/TCF inhibitor FH535. (5) Rapamycin blocks TORC1dependent translation while cycloheximide inhibits global translation. Note that some proteins are regulated simultaneously by more than one Wnt/LRP6 signaling cascade, for instance cyclin D1 and Myc ([1_TD$IF]see Table 1 [12_TD$IF]in [13_TD$IF]main text).
1. Wnt response, inhibited by Dkk1?
YES
2. Inducible with BIO?
YES 3. Protein levels increase with BIO or Wnt3a?
YES 4. Modulated by β-cat?
NO 5. Wnt response, inhibited by rapamycin or cycloheximide?
NO
NO
Wnt/PCP or Wnt/Ca pathway
Upstream Wnt/LRP6 cascade
NO
YES
Determine the Wnt/β-catenin modulaon of pathway target acvity
YES Wnt/TOR pathway
NO Wnt/STOP pathway
Figure I. A Practical Guide to Branches of Wnt/LRP6 Signaling.
b-transducin repeat-containing protein E3 ubiquitin ligase (b-TrCP) (reviewed in [20]). In the absence of Wnt ligands, CK1/ and GSK3 phosphorylate b-catenin in the destruction complex, targeting it for b-TrCP-mediated proteasomal degradation [21]. Wnt/LRP6 signaling inhibits the destruction complex by at least two mechanisms. First, GSK3 becomes product-inhibited by phospho-LRP6 [22]. Second, signalosomes mature into multivesicular bodies where the destruction complex is sequestered together with LRP6 [23–25] (Figure 1B). Wnt/LRP6 signaling can be enhanced by members of the R-spondin (RSPO) family of secreted proteins [26], which bind to LGR4/5 receptors and the E3 protein ligases RNF43/ZNRF3 to stabilize the Wnt receptors at the cell surface [27–29].
Wnt/b-Catenin Signaling At the heart of canonical Wnt signaling is the transcriptional regulator b-catenin which, in the absence of Wnt ligands, is targeted for proteasomal degradation [21]. Wnt/LRP6 signaling inhibits GSK3, which allows newly synthesized b-catenin to accumulate in the cytoplasm and translocate to the nucleus where it binds members of the T cell factor/lymphocyte enhancer factor family (TCF/LEF) to initiate transcription of target genes [21,30,31]. The transcriptional program regulated by Wnt/b-catenin signaling depends on the cellular context, but it usually directs proliferation, survival, and inhibition of differentiation (reviewed in [32]). The best-established direct
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Wnt/b-catenin target gene is Axin2, a negative feedback regulator whose expression is typically an indicator of active Wnt/b-catenin signaling [1,33]. Many other Wnt/b-catenin target genes have been identified (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/), including Myc [34], which promotes proliferation, especially in stem cells. In colorectal cancer, truncating mutations of APC lead to the upregulation of Myc, which promotes neoplasia [35].
Wnt-Dependent Stabilization of Proteins (Wnt/STOP) GSK3 is a promiscuous kinase that phosphorylates many other proteins in addition to b-catenin [36]. Phosphorylation by GSK3 can generate phospho-degrons that are recognized by dedicated E3 ubiquitin ligases, notably b-TrCP, FBXW7, and NEDD4L, which target these substrates for proteasomal degradation [36–40]. Wnt/LRP6 signaling can sequester up to 70% of cellular GSK3 activity in multivesicular bodies, as well as a large proportion of the cellular ubiquitin pool, thereby stabilizing many other proteins in addition to b-catenin [24,25,41]. As a consequence, the half-life of total cellular proteins increases from 9 to 12 h upon Wnt treatment in HEK293T cells [24]. Indeed, it has been recognized that several dozen proteins beyond b-catenin are bona fide GSK3 targets and are regulated by Wnt [18,24,36,42]. This[15_TD$IF] process, which is now referred as Wnt-dependent stabilization of proteins [16_TD$IF](Wnt/STOP[17_TD$IF]), peaks during mitosis to slow down protein degradation as cells prepare to divide [42]. Wnt/STOP targets are involved in many cellular processes including cell-cycle progression, endolysosomal biogenesis, crosstalk with other pathways, DNA remodeling, and the cytoskeleton (Table 1).
Other b-Catenin-Independent Cascades of Wnt/LRP6 Signaling
In addition to Wnt/STOP, other b-catenin-independent Wnt/LRP6 signaling cascades branch off at GSK3, which not only targets proteins to degradation but can also modulate their activity by phosphorylation. GSK3 inhibition by Wnt/LRP6 signaling activates target of rapamycin (TOR) to increase protein translation [43], modulates the activity of microtubule-associated proteins (reviewed in [44]), and inhibits the activity of protein phosphatase 1 (PP1) [18]. Wnt/LRP6 signaling also regulates localization and clustering of APC, Axin, and Dvl with components of the cytoskeleton during spindle orientation, centrosome organization, cell movement, and axonal growth [45–47]. We review below the different roles of b-catenin-independent Wnt/LRP6 signaling as well as the molecular mechanisms underlying them.
Roles of b-Catenin-Independent Wnt/LRP6 Signaling Cell Growth Cells need to synchronize cell-cycle progression with cell growth to maintain a constant cell size along their lineages. To achieve this, mitogenic pathways such as Wnt/LRP6 signaling coregulate anabolic/catabolic cascades and cell-cycle effectors to maintain proliferation [48]. GSK3 is a key regulator of cellular catabolism, and inhibits cell growth by blocking glucose intake, reduces TORmediated protein translation, and destabilizes key cell-cycle effectors [49–51]. Thus, by inhibiting GSK3 activity, Wnt/LRP6 signaling can impact both on growth and cell-cycle progression. In the absence of Wnt ligands, GSK3 phosphorylates tuberous sclerosis complex 2 (TSC2), which promotes its binding to and inhibition of TOR complex 1 (TORC1). Wnt/LRP6 signaling blocks phosphorylation of TSC2 and thereby activates the TOR pathway to stimulate protein translation during G1 [43]. Wnt-dependent activation of TOR also increases the translation of cyclin D1 mRNA [43], and thereby couples growth and cell-cycle progression through G1. Wnt/LRP6 signaling not only impacts on cell growth through GSK3 by increasing protein translation but also by decreasing protein degradation. Around 15% of the cellular proteins are stabilized by Wnt/STOP signaling as cells prepare to divide, and this provides a growth advantage to the daughter cells [41,42]. Wnt/STOP signaling increases the size of various cell
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Table 1. Wnt/LRP6 Target Proteinsa Stabilized by Wnt/STOP
ATOH1
[96]
BRD3
[18]
BUB1
[18]
CCND1
[42]
[98]
CDC25A
[42]
[99]
CENPT
[42]
CJUN; BJUN
[24]
CTNNB1
[101–103]
CTNNG
[103,104]
FOXM1
[105]
HDAC4
[24]
HDAC7
[42]
HINT2
[42]
MAP1B; MAP2
[66]
[66,106]
MAPT
[107]
[108,109]
MARK3
[42]
MITF
[71]
MRLC
[111]
MYC
[42]
PLK1
[17]
PPP1R2
Activity Modulated by Wnt/LRP6 [6_TD$IF]Signaling
Transcriptionally [7_TD$IF]Induced by Wnt/b-[8_TD$IF]Catenin
Protein ID
[97]
[100]
[110]
[34]
[18]
RPSA
[42]
SEPT4
[18]
SAM68
[111]
SMAD1
[112]
SMAD3
[113]
SMAD4
[81]
SNAIL
[114]
SRC3
[116]
SREBP1
[117]
YAP/TAZ
[83,84]
TESC
[111]
TMEM4
[111]
TP53
[118]
TSC2
[18]
[115]
[43]
UBE2 C
[17]
VENTX
[111]
[119]
a
For simplicity, only proteins that are post-transcriptionally regulated by Wnt/Lrp6 signaling are shown. Human official symbols are indicated regardless of the species analyzed in the referenced studies.
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types during G1 by up to 25%, independently of TOR activation and b-catenin. Beyond increasing bulk protein, Wnt/STOP also stabilizes the G1 activators cyclin D1 and Myc, which may couple growth and cell-cycle progression in daughter cells [41,42]. Mitotic Wnt/LRP6 Signaling A conserved feature of Wnt/LRP6 signaling is that the pathway peaks at mitosis as a result of CCNY-dependent phosphorylation of LRP6 [16]. During mitosis cells mechanically segregate cellular components into daughter cells, while transcription and translation remain mostly silent. Evidence is accumulating that transcription-independent Wnt/LRP6 signaling orchestrates a rich mitotic program. Wnts have evolutionary conserved roles in regulating the orientation of cell divisions [46,52–54]. In mouse embryonic stem cells (mESCs), oriented Wnt3a ligands trigger mitotic Wnt/LRP6 signaling to align the spindle and to asymmetrically segregate cellular components into daughter cells [54]. The Wnt-proximal daughter cell retains the older centriole, retains pluripotencyassociated proteins (e.g., Sox2), and inherits most of the cellular components of the Wnt/ LRP6 signaling pathway [54]. Because Wnts are short-range signals, polarized Wnt/LRP6 signaling in stem cell niches may regulate cell fate by asymmetrically segregating cellular determinants into daughter cells. Consistent with this hypothesis, asymmetric cell division of intestinal stem cells is regulated by Wnt-secreting Paneth cells. Upon cell division the Wntproximal daughter cell maintains stem cell fate and inherits the unreplicated DNA [55]. According to Cairns’ hypothesis, this may minimize mutations in stem cells introduced by replication errors [56]. It is still not well understood what molecular mechanisms underlie Wnt/LRP6 signaling effects on mitotic spindle orientation in stem cells. Studies in HeLa cells have shown that Dvl cooperates with Polo-like kinase 1 (PLK1) in establishing spindle orientation, which can be regulated by Wnt/LRP6 signaling [46]. In addition, APC associates with the microtubule plus-ends and connects them to kinetochores, which is essential for chromosome organization and segregation [57,58]. However, it remains unclear whether APC functions in Wnt/ LRP6 signaling solely through its role in the destruction complex or also via microtubuleassociated mechanisms. Moreover, GSK3 associates with centrosomes where it can regulate microtubule growth [59]. This is regulated by Wnt/STOP signaling, which is crucial for faithful chromosome segregation during mitosis [60]. Depletion of LRP6 increases microtubule assembly rates, which can be rescued by depletion of APC and Axin, but not of b-catenin. As a consequence, inhibition of Wnt/STOP over several generations leads to aneuploidy [60,61]. The Wnt/STOP targets that modulate the assembly rates of microtubules are unknown. Candidates include Wnt/STOP targets such as PLK1, ubiquitin-conjugating enzyme E2 C (UBE2 C/UBCH10), microtubule-associated protein 1B (MAP1B), MAP2, and Tau (MAPT) (Table 1). Consistent with its important roles in mitosis, misregulation of components of Wnt/LRP6 signaling in vivo leads to severe mitotic phenotypes. Notably, truncating APC mutations randomize the division plane of intestinal stem cells and block the asymmetric distribution of DNA strands into daughter cells, thereby perturbing tissue architecture [55]. Eventually APC truncations induce chromosome instability and contribute to tumorigenesis [57]. Wnt/LRP6 Signaling in Germ Cells Studying the physiological role of transcription-independent Wnt/LRP6 signaling in vivo is challenging given the pervasive roles of b-catenin [62]. However, germ cells are an exception because, during the last steps of their maturation, both oocytes and spermatozoa are transcriptionally silent, and b-catenin becomes dispensable [17,63].
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+ – p-Lrp6 –
+ Wnt exosomes
Figure 2. Wnt/LRP6 Signaling Functions in Transcriptionally-Silent Sperm. Immunostaining of phosphorylated LRP6 (p-LRP6; Tp1479) in mouse sperm. P-LRP6 marks activated Wnt/ LRP6 signaling, is largely restricted to the midpiece of sperm, and is superinduced by Wnt-containing exosomes released from the epididymis. DNA is shown in green.
[2_TD$IF]Wnt/STOP signaling functions in mature Xenopus oocytes to promote the first embryonic cell divisions [17]. Depleting Xenopus oocytes of maternal Lrp6 or Ccny, but not of b-catenin, leads to cell-cycle arrest at the one- or two-cell embryonic stage in a GSK3-dependent manner. This is because Wnt/STOP signaling in oocytes is necessary to stabilize mitotic effectors, including Plk1 and Ube2c. Likewise, Wnt/LRP6 signaling is required for maturation of transcriptionally-silent sperm [18]. Mice mutant for the Wnt/LRP6 signaling component Ccnyl1 are male-infertile and harbor immotile sperm [18,64,65]. Testicular sperm passing through the epididymis receive signals to terminally differentiate and to gain motility before ejaculation. The epididymal epithelium expresses [18_TD$IF]several [19_TD$IF]Wnts including Wnt2b, which is secreted in exosomes to the lumen. Wnt/ LRP6 signaling functions in sperm (Figure 2), where it elicits at least three b-catenin-independent responses via GSK3 repression [18,64]: first, it induces Wnt/STOP signaling to maintain protein homeostasis. Second, it establishes the membrane diffusion barrier in the sperm tail by promoting the clustering at the annulus of septin 4 (Sept4), a ring-forming protein controlling membrane dynamics. Finally, it initiates sperm motility by inhibiting PP1 phosphatase activity via reducing phosphorylation and binding of its inhibitory subunit PPP1R2. Consistently, overexpression of DKK1 in the epididymis reduces sperm motility, while truncated APC (APCmin[14_TD$IF]) increases it [18]. Importantly, the factors targeted by Wnt/LRP6 signaling in oocytes and sperm, including Sept4, PP1, and PLK1, play crucial roles in somatic cells as well, especially during mitosis. Thus, the lessons learned in germ cells may have broader relevance. Wnt/LRP6 Signaling in Neurons Neuronal connections are not only created de novo during development but are rewired in adult neural networks in an activity-dependent manner. The development and plasticity of synapses are often post-transcriptionally regulated in view of the long distances between synaptic terminals and the nucleus. Wnt/LRP6 signaling has emerged as a key regulator of axonal remodeling, synaptic plasticity, neurite outgrowth, and neurotransmitter release independently of b-catenin. In mouse cerebellum, Wnt7a localizes in synaptic areas, where it increases the size and spreading of the axonal growth cone [45,66]. Moreover, depletion of Wnt7a and Dvl1 impairs neurotransmitter release in mouse cerebellum [67–69]. Several lines of evidence suggest that these processes are modulated by Wnt/LRP6 signaling acting directly on the cytoskeleton. GSK3 phosphorylates the microtubule-associated proteins Tau, MAP1B, and MAP2, thereby destabilizing the microtubules at synaptic terminals [45,66]. Wnts reduce MAP1B phosphorylation, stabilize the microtubules, and induce axonal remodeling by inhibiting GSK3, independently of b-catenin or transcription [47,68]. Consistently, chemical inhibition of GSK3 mimics the Wnt7a remodeling effect in axons [47,66,68–70]. Likewise, Wnt/STOP signaling stabilizes Tau in non-neuronal cells [71].
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Tau phosphorylation and stability play crucial roles in axon guidance and maintenance, and its misregulation is a hallmark of some neurodegenerative disorders, notably Alzheimer [72,73]. In addition, LRP6 mutations are associated with late-onset Alzheimer's disease [74]. Thus, it appears promising to probe into the role of b-catenin-independent Wnt/LRP6 signaling in these disorders. Wnts also stimulate neurite outgrowth, and accumulating evidence suggests that this effect is mediated via Wnt/LRP6 signaling. First, chemical inhibition of GSK3 mimics Wnt-dependent neurite outgrowth, while Axin overexpression blocks it [47,66,75]. Second, APC localizes at the tip of hippocampal neurites with unphosphorylated Tau [76]. Third, although b-catenin plays structural roles in the neurite, its transcriptional activity is dispensable for neurite outgrowth [75,76]. Roles of b-catenin-independent Wnt/LRP6 signaling in neurons are evolutionarily conserved. In Drosophila, Wg (Wnt) inhibits Shaggy (GSK3) to stabilize synaptic microtubules through changes in the localization of Futsch (MAP1B) [77,78]. In addition, presynaptic Wg, Arrow (LRP6), and Shaggy, but not Armadillo (b-catenin), are required for neuromuscular junction development in Drosophila [78]. Similarly, Wg and Shaggy, but not Pangolin (TCF) or Armadillo, promote neuronal stability in the adult olfactory system [79]. In light of Wnt/STOP signaling, it appears fruitful to investigate which Shaggy/GSK3 target proteins modulate these effects in fly.
Crosstalk with other Signaling Cascades Cells typically receive multiple signals, and particular components act as signaling hubs that coordinate crosstalk between cascades. GSK3 is a key example of a signaling hub which not only integrates several inputs, such as Wnt, EGF, and insulin, but also multiple outputs (e.g., Table 1), thereby allowing Wnt/LRP6 signaling to directly regulate other pathways independently of b-catenin. During vertebrate axial patterning, the dorsal–ventral and anterior–posterior axes are regulated by BMP and Wnt gradients, respectively [80]. A key effector of the BMP pathway is Smad1, which is a Wnt/STOP target [38]. By stabilizing Smad1, Wnt/STOP signaling increases the duration of BMP signaling both in vitro and in Xenopus embryos, where it promotes the formation of the epidermis. Similarly, Wnt/STOP signaling regulates the stability of Smad4 [81,82], allowing Wnt ligands to enhance TGF-b signaling, but only in the presence of FGF. This is because GSK3dependent degradation of Smad4 requires priming phosphorylation by MAPK, which is activated by FGF. In the absence of FGF ligands, Wnt and TGF-b signaling remain insulated because GSK3 cannot phosphorylate the Smad4 degron [81]. Mutation of these sites inhibits germ-layer specification and Spemann–Mangold organizer formation during Xenopus development, highlighting the importance of this integrative network. Finally, Wnt/LRP6 signaling activates the Hippo pathway by stabilizing its transducer YAP/TAZ and inducing its nuclear translocation to promote differentiation of mesenchymal stem cells, among other processes [83,84]. Interestingly, YAP/TAZ accounts for a significant part of the Wnt transcriptional program, and is required in vivo for intestinal crypt overgrowth induced by APC deficiency [83].
Concluding Remarks and Future Directions The discovery of b-catenin-independent Wnt/LRP6 signaling branches raises the important question of the extent to which Wnt responses are mediated by transcriptional or b-cateninindependent routes (see Outstanding Questions). At the one extreme, during mouse gastrulation and Drosophila segmentation, Wnt3/Wg mutants phenocopy b-catenin/armadillo mutants, suggesting that transcriptional regulation is the dominant signaling mode [85–87]. At the other
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Outstanding Questions What are the post-transcriptional protein targets for Wnt/LRP6 signaling, notably in mitotic cells? Given the various other signaling inputs into GSK3 (e.g., insulin and EGF) and the large number of GSK3 substrates, what provides the specificity for different Wnt/LRP6 outputs? In which other biological processes does transcription-independent Wnt/ LRP6 signaling play a role? Does misregulation of b-catenin-independent Wnt/LRP6 signaling contribute to Wnt-associated diseases such as colorectal cancer?
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extreme, in transcriptionally-silent mouse sperm and frog eggs only b-catenin-independent Wnt signaling occurs. Even in HeLa cells, a 3 h Wnt3a pulse only induces the expression of three genes, a minor effect compared with the stabilization of 15% of the proteome through Wnt/ STOP, the modulation of the orientation of the mitotic spindle, and the upregulation of translation through the TOR pathway [42,43,46]. It is likely that other cell types will lie between these extremes and engage both b-catenin-dependent and -independent Wnt/LRP6 signaling branches. In dividing cells, a common theme among Wnt/LRP6-triggered signaling branches is that they all seem to orchestrate a growth program, for example by stimulating protein production via (i) increased gene expression (Wnt/b-catenin), (ii) enhanced protein biosynthesis (Wnt/TOR), and (iii) reduced protein degradation (Wnt/STOP). Another question is, given the various signaling inputs into GSK3 (e.g., insulin and EGF), what provides the specificity for the Wnt/LRP6 outputs? It has been proposed that only those GSK3 substrates that bind to the destruction complex, such as b-catenin and YAP/TAZ, can be targeted by Wnt ligands [88]. However, Wnt/LRP6 signaling depletes up to 70% of cytoplasmic GSK3 activity in multivesicular bodies, and this sequestration itself requires Axin [24,25,41]. Consistent with this, Wnt/LRP6 modulation of GSK3 targets other than b-catenin, such as YAP/ TAZ, Snail, or CNPY2, is also Axin/APC-dependent (Table 1). Thus, additional specificity may be provided by dedicated priming kinases for GSK3, as well as E3 ubiquitin ligases and their respective regulators. For instance, the TGFb effector Smad4 is only stabilized by Wnt/STOP signaling if gated by FGF, which activates MAPK and primes for GSK3, thus rendering Smad4 Wnt-sensitive [81]. Conversely, regulation of particular GSK3 targets, such as MAP1B, is shared between Wnt and EGF signaling [89]. Finally, the exploration of b-catenin-independent Wnt/LRP6 cascades may open novel options for the treatment of Wnt-associated diseases. Currently the pharmaceutical industry focuses on targeting the Wnt/b-catenin branch. However, as discussed here, misregulation of b-cateninindependent Wnt/LRP6 signaling affects cell growth and proliferation, as well as genome stability, which are hallmarks of cancer cells. Thus, there may be novel opportunities for pharmacological intervention with colorectal cancers harboring mutations upstream of GSK3 [28,90–94] by blocking b-catenin-independent Wnt/LRP6 branches. Acknowledgments We apologize to all authors whose primary work could not be cited owing to space constraints or whose work was inadvertently overlooked. This work was supported by the Deutsche Forschungsgemeinschaft.
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Review
b-Catenin-Independent Roles of Wnt/LRP6 Signaling Sergio P. Acebron1,* and Christof Niehrs1,2,* Wnt/LRP6 signaling is best known for the b-catenin-dependent regulation of target genes. However, pathway branches have recently emerged, including Wnt/STOP signaling, which act independently of b-catenin and transcription. We review here the molecular mechanisms underlying b-catenin-independent Wnt/LRP6 signaling cascades and their implications for cell biology, development, and physiology.
Wnt/LRP6 Signaling Wnt proteins are secreted short-range signals found in all metazoans, which function in embryonic development and adult tissue homeostasis by regulating fate, proliferation, polarity, and migration of cells [1–3]. Misregulation of Wnt signaling can lead to disease, notably cancer [4,5]. In most mammals there are 19 Wnt genes that can signal through a variety of receptors and downstream pathways (reviewed in [6]). Roughly, Wnt signaling is subdivided into a canonical pathway, also known as the Wnt/b-catenin cascade, and a non-canonical pathway, the most prominent of which is the Wnt/PCP (planar cell polarity) pathway (reviewed in [7]). Most cellular Wnt signaling is transmitted via seven transmembrane receptors of the Frizzled (Fzd) family [6]. A characteristic feature of Wnt/b-catenin signaling is that it not only involves Fzd receptors but additionally the Wnt coreceptors lipoprotein receptor-related proteins 5,6 (LRP5 and LRP6). Wnt binding to Fzd/LRP6 activates what has traditionally been viewed as a linear signal transduction cascade, which culminates in the stabilization of b-catenin. However, it has become clear that this pathway branches off to trigger a variety of other processes which are independent of b-catenin-mediated transcription (Figure 1A and Box 1). The term Wnt/LRP6 signaling encompasses all these downstream events, including but not limited to Wnt/b-catenin signaling, also known as canonical Wnt signaling. We review here the different b-catenin-independent events triggered by Wnt/LRP6 signaling and their role in cell biology, development, and physiology. Reviews focused on non-canonical Wnt signaling can be found elsewhere [3,8,9].
Trends Wnt/LRP6 signaling is thought to proceed by b-catenin mediated transcription. However, this pathway branches off to trigger a variety of other processes, revising the traditional transcription-centric view. Wnt/STOP signaling is one such novel branch of Wnt/LRP6 signaling, which peaks during mitosis to slow down protein degradation as cells prepare to divide. Transcription-independent Wnt/LRP6 signaling regulates cell growth and mitotic progression in dividing cells, axonal remodeling in post-mitotic neurons, and maturation of germ cells.
The Upstream Cascade Upon Wnt ligand binding, Fzd/LRP6 receptors cluster together with the scaffold protein Dishevelled (Dvl) into signalosomes, which are endocytosed [10–12] (Figure 1B). Signalosome formation induces LRP6 phosphorylation by glycogen synthase kinase 3 (GSK3) at PPPSP motifs, which primes adjacent S/T clusters for phosphorylation by casein kinase 1g (CK1g) [13,14]. In addition to GSK3, other kinases phosphorylate the LRP6 PPPSP motifs in a Wnt-independent manner (reviewed in [15]). For instance, the competence of LRP6 to respond to Wnt ligands is maximal during G2/M owing to primed phosphorylation by cyclin-dependent kinase 14 (CDK14/ PFTK1) and its regulators, mitotic cyclin Y and cyclin Y-like 1 (CCNY/CCNYL1) [16–19]. Phosphorylated LRP6 recruits a multiprotein ‘destruction complex’ that contains the scaffold proteins Axin and adenomatous polyposis coli (APC), the kinases CK1/ and GSK3, and the
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1 Division of Molecular Embryology, Deutsches Krebsforschungszentrum (DKFZ)–Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH) Alliance, 69120 Heidelberg, Germany 2 Institute of Molecular Biology, 55128 Mainz, Germany
*Correspondence: [email protected] (S.P. Acebron) and [email protected] (C. Niehrs).
http://dx.doi.org/10.1016/j.tcb.2016.07.009 © 2016 Elsevier Ltd. All rights reserved.
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(A)
Wnts ‘Non-canonical’ Wnt/PCP, Wnt/Ca+2
Wnt/LRP6 signaling ‘Canonical’
β-catenin independent signaling
Wnt/β-catenin signaling
Wnt/STOP signaling
Other: e.g., Wnt/TOR
(B) Wnt
Fzd
LRP6 DvI Signalosome
MVB
β-catenin destrucon complex GSK3
Wnt/β-catenin signaling GSK3
Wnt/STOP signaling
GSK3 SK
GSK3 SK
20 S
β-Cat β-Cat
Wnt/TOR signaling
TSC2
P
20 S
TSC2 Protein stabilizaon
β-Cat
Ribosome β-Cat
Gene transcripon
TORC1
TCF
Protein translaon
Cell growth Cell division
Figure 1. Wnt Signaling Pathways. (A) Hierarchy of different Wnt signaling cascades. (B) Model of the Wnt/LRP6 signaling pathway. Wnt ligands form a ternary complex with LRP6 and Frizzled[3_TD$IF] (Fzd), which cluster on platforms of Dvl. These clusters are internalized in signalosomes, and recruit the destruction complex. LRP6 signalosomes further mature into multivesicular bodies where they sequester GSK3. Wnt/LRP6 signaling triggers several downstream cascades. In Wnt/ b-catenin signaling it leads to the stabilization of b-catenin (b-Cat), which upregulates the transcription of target genes. In Wnt/TOR signaling it blocks GSK3-dependent phosphorylation of TSC2, thereby derepressing TORC1 and promoting protein translation. In Wnt/STOP signaling it stabilizes GSK3 targets which are required for example for cell growth and division. Note that Wnt/LRP6 signaling can modulate the activity of other GSK3 targets in addition to TSC2 (Table 1[4_TD$IF]) Proteasome (20 S).
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Box 1. A Practical Guide to Branches of Wnt/LRP6 Schematic of experimental flow (1–5) to assess involvement of specific downstream cascades in a Wnt-induced response[9_TD$IF] (Figure I). Note that these assays serve only as a guideline for first analysis and that full assignment to a given cascade may require further ongoing investigations for the directness of effects. For individual examples see Table [10_TD$IF]1 in main text. (1) Wnt3a is well established in Wnt/LRP6 signaling and the recombinant protein is commercially available, or can be used as conditioned medium. Dkk1 inhibits Wnt/LRP6 signaling [95], and is commercially available. (2) 6Bromoindirubin-3’-oxime (BIO) is a chemical inhibitor of GSK3. (3) GSK3 targets that do not show increased protein levels upon Wnt3a or BIO may be directly activity-modulated. Phosphorylation status can be analyzed, for example by phosphatase- and BIO-sensitive mobility shift in 1D or 2D gels. (4) Dependency on b-catenin can be analyzed by overexpression or depletion of b-catenin, or by using the b-catenin/TCF inhibitor FH535. (5) Rapamycin blocks TORC1dependent translation while cycloheximide inhibits global translation. Note that some proteins are regulated simultaneously by more than one Wnt/LRP6 signaling cascade, for instance cyclin D1 and Myc ([1_TD$IF]see Table 1 [12_TD$IF]in [13_TD$IF]main text).
1. Wnt response, inhibited by Dkk1?
YES
2. Inducible with BIO?
YES 3. Protein levels increase with BIO or Wnt3a?
YES 4. Modulated by β-cat?
NO 5. Wnt response, inhibited by rapamycin or cycloheximide?
NO
NO
Wnt/PCP or Wnt/Ca pathway
Upstream Wnt/LRP6 cascade
NO
YES
Determine the Wnt/β-catenin modulaon of pathway target acvity
YES Wnt/TOR pathway
NO Wnt/STOP pathway
Figure I. A Practical Guide to Branches of Wnt/LRP6 Signaling.
b-transducin repeat-containing protein E3 ubiquitin ligase (b-TrCP) (reviewed in [20]). In the absence of Wnt ligands, CK1/ and GSK3 phosphorylate b-catenin in the destruction complex, targeting it for b-TrCP-mediated proteasomal degradation [21]. Wnt/LRP6 signaling inhibits the destruction complex by at least two mechanisms. First, GSK3 becomes product-inhibited by phospho-LRP6 [22]. Second, signalosomes mature into multivesicular bodies where the destruction complex is sequestered together with LRP6 [23–25] (Figure 1B). Wnt/LRP6 signaling can be enhanced by members of the R-spondin (RSPO) family of secreted proteins [26], which bind to LGR4/5 receptors and the E3 protein ligases RNF43/ZNRF3 to stabilize the Wnt receptors at the cell surface [27–29].
Wnt/b-Catenin Signaling At the heart of canonical Wnt signaling is the transcriptional regulator b-catenin which, in the absence of Wnt ligands, is targeted for proteasomal degradation [21]. Wnt/LRP6 signaling inhibits GSK3, which allows newly synthesized b-catenin to accumulate in the cytoplasm and translocate to the nucleus where it binds members of the T cell factor/lymphocyte enhancer factor family (TCF/LEF) to initiate transcription of target genes [21,30,31]. The transcriptional program regulated by Wnt/b-catenin signaling depends on the cellular context, but it usually directs proliferation, survival, and inhibition of differentiation (reviewed in [32]). The best-established direct
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Wnt/b-catenin target gene is Axin2, a negative feedback regulator whose expression is typically an indicator of active Wnt/b-catenin signaling [1,33]. Many other Wnt/b-catenin target genes have been identified (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/), including Myc [34], which promotes proliferation, especially in stem cells. In colorectal cancer, truncating mutations of APC lead to the upregulation of Myc, which promotes neoplasia [35].
Wnt-Dependent Stabilization of Proteins (Wnt/STOP) GSK3 is a promiscuous kinase that phosphorylates many other proteins in addition to b-catenin [36]. Phosphorylation by GSK3 can generate phospho-degrons that are recognized by dedicated E3 ubiquitin ligases, notably b-TrCP, FBXW7, and NEDD4L, which target these substrates for proteasomal degradation [36–40]. Wnt/LRP6 signaling can sequester up to 70% of cellular GSK3 activity in multivesicular bodies, as well as a large proportion of the cellular ubiquitin pool, thereby stabilizing many other proteins in addition to b-catenin [24,25,41]. As a consequence, the half-life of total cellular proteins increases from 9 to 12 h upon Wnt treatment in HEK293T cells [24]. Indeed, it has been recognized that several dozen proteins beyond b-catenin are bona fide GSK3 targets and are regulated by Wnt [18,24,36,42]. This[15_TD$IF] process, which is now referred as Wnt-dependent stabilization of proteins [16_TD$IF](Wnt/STOP[17_TD$IF]), peaks during mitosis to slow down protein degradation as cells prepare to divide [42]. Wnt/STOP targets are involved in many cellular processes including cell-cycle progression, endolysosomal biogenesis, crosstalk with other pathways, DNA remodeling, and the cytoskeleton (Table 1).
Other b-Catenin-Independent Cascades of Wnt/LRP6 Signaling
In addition to Wnt/STOP, other b-catenin-independent Wnt/LRP6 signaling cascades branch off at GSK3, which not only targets proteins to degradation but can also modulate their activity by phosphorylation. GSK3 inhibition by Wnt/LRP6 signaling activates target of rapamycin (TOR) to increase protein translation [43], modulates the activity of microtubule-associated proteins (reviewed in [44]), and inhibits the activity of protein phosphatase 1 (PP1) [18]. Wnt/LRP6 signaling also regulates localization and clustering of APC, Axin, and Dvl with components of the cytoskeleton during spindle orientation, centrosome organization, cell movement, and axonal growth [45–47]. We review below the different roles of b-catenin-independent Wnt/LRP6 signaling as well as the molecular mechanisms underlying them.
Roles of b-Catenin-Independent Wnt/LRP6 Signaling Cell Growth Cells need to synchronize cell-cycle progression with cell growth to maintain a constant cell size along their lineages. To achieve this, mitogenic pathways such as Wnt/LRP6 signaling coregulate anabolic/catabolic cascades and cell-cycle effectors to maintain proliferation [48]. GSK3 is a key regulator of cellular catabolism, and inhibits cell growth by blocking glucose intake, reduces TORmediated protein translation, and destabilizes key cell-cycle effectors [49–51]. Thus, by inhibiting GSK3 activity, Wnt/LRP6 signaling can impact both on growth and cell-cycle progression. In the absence of Wnt ligands, GSK3 phosphorylates tuberous sclerosis complex 2 (TSC2), which promotes its binding to and inhibition of TOR complex 1 (TORC1). Wnt/LRP6 signaling blocks phosphorylation of TSC2 and thereby activates the TOR pathway to stimulate protein translation during G1 [43]. Wnt-dependent activation of TOR also increases the translation of cyclin D1 mRNA [43], and thereby couples growth and cell-cycle progression through G1. Wnt/LRP6 signaling not only impacts on cell growth through GSK3 by increasing protein translation but also by decreasing protein degradation. Around 15% of the cellular proteins are stabilized by Wnt/STOP signaling as cells prepare to divide, and this provides a growth advantage to the daughter cells [41,42]. Wnt/STOP signaling increases the size of various cell
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Table 1. Wnt/LRP6 Target Proteinsa Stabilized by Wnt/STOP
ATOH1
[96]
BRD3
[18]
BUB1
[18]
CCND1
[42]
[98]
CDC25A
[42]
[99]
CENPT
[42]
CJUN; BJUN
[24]
CTNNB1
[101–103]
CTNNG
[103,104]
FOXM1
[105]
HDAC4
[24]
HDAC7
[42]
HINT2
[42]
MAP1B; MAP2
[66]
[66,106]
MAPT
[107]
[108,109]
MARK3
[42]
MITF
[71]
MRLC
[111]
MYC
[42]
PLK1
[17]
PPP1R2
Activity Modulated by Wnt/LRP6 [6_TD$IF]Signaling
Transcriptionally [7_TD$IF]Induced by Wnt/b-[8_TD$IF]Catenin
Protein ID
[97]
[100]
[110]
[34]
[18]
RPSA
[42]
SEPT4
[18]
SAM68
[111]
SMAD1
[112]
SMAD3
[113]
SMAD4
[81]
SNAIL
[114]
SRC3
[116]
SREBP1
[117]
YAP/TAZ
[83,84]
TESC
[111]
TMEM4
[111]
TP53
[118]
TSC2
[18]
[115]
[43]
UBE2 C
[17]
VENTX
[111]
[119]
a
For simplicity, only proteins that are post-transcriptionally regulated by Wnt/Lrp6 signaling are shown. Human official symbols are indicated regardless of the species analyzed in the referenced studies.
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types during G1 by up to 25%, independently of TOR activation and b-catenin. Beyond increasing bulk protein, Wnt/STOP also stabilizes the G1 activators cyclin D1 and Myc, which may couple growth and cell-cycle progression in daughter cells [41,42]. Mitotic Wnt/LRP6 Signaling A conserved feature of Wnt/LRP6 signaling is that the pathway peaks at mitosis as a result of CCNY-dependent phosphorylation of LRP6 [16]. During mitosis cells mechanically segregate cellular components into daughter cells, while transcription and translation remain mostly silent. Evidence is accumulating that transcription-independent Wnt/LRP6 signaling orchestrates a rich mitotic program. Wnts have evolutionary conserved roles in regulating the orientation of cell divisions [46,52–54]. In mouse embryonic stem cells (mESCs), oriented Wnt3a ligands trigger mitotic Wnt/LRP6 signaling to align the spindle and to asymmetrically segregate cellular components into daughter cells [54]. The Wnt-proximal daughter cell retains the older centriole, retains pluripotencyassociated proteins (e.g., Sox2), and inherits most of the cellular components of the Wnt/ LRP6 signaling pathway [54]. Because Wnts are short-range signals, polarized Wnt/LRP6 signaling in stem cell niches may regulate cell fate by asymmetrically segregating cellular determinants into daughter cells. Consistent with this hypothesis, asymmetric cell division of intestinal stem cells is regulated by Wnt-secreting Paneth cells. Upon cell division the Wntproximal daughter cell maintains stem cell fate and inherits the unreplicated DNA [55]. According to Cairns’ hypothesis, this may minimize mutations in stem cells introduced by replication errors [56]. It is still not well understood what molecular mechanisms underlie Wnt/LRP6 signaling effects on mitotic spindle orientation in stem cells. Studies in HeLa cells have shown that Dvl cooperates with Polo-like kinase 1 (PLK1) in establishing spindle orientation, which can be regulated by Wnt/LRP6 signaling [46]. In addition, APC associates with the microtubule plus-ends and connects them to kinetochores, which is essential for chromosome organization and segregation [57,58]. However, it remains unclear whether APC functions in Wnt/ LRP6 signaling solely through its role in the destruction complex or also via microtubuleassociated mechanisms. Moreover, GSK3 associates with centrosomes where it can regulate microtubule growth [59]. This is regulated by Wnt/STOP signaling, which is crucial for faithful chromosome segregation during mitosis [60]. Depletion of LRP6 increases microtubule assembly rates, which can be rescued by depletion of APC and Axin, but not of b-catenin. As a consequence, inhibition of Wnt/STOP over several generations leads to aneuploidy [60,61]. The Wnt/STOP targets that modulate the assembly rates of microtubules are unknown. Candidates include Wnt/STOP targets such as PLK1, ubiquitin-conjugating enzyme E2 C (UBE2 C/UBCH10), microtubule-associated protein 1B (MAP1B), MAP2, and Tau (MAPT) (Table 1). Consistent with its important roles in mitosis, misregulation of components of Wnt/LRP6 signaling in vivo leads to severe mitotic phenotypes. Notably, truncating APC mutations randomize the division plane of intestinal stem cells and block the asymmetric distribution of DNA strands into daughter cells, thereby perturbing tissue architecture [55]. Eventually APC truncations induce chromosome instability and contribute to tumorigenesis [57]. Wnt/LRP6 Signaling in Germ Cells Studying the physiological role of transcription-independent Wnt/LRP6 signaling in vivo is challenging given the pervasive roles of b-catenin [62]. However, germ cells are an exception because, during the last steps of their maturation, both oocytes and spermatozoa are transcriptionally silent, and b-catenin becomes dispensable [17,63].
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+ – p-Lrp6 –
+ Wnt exosomes
Figure 2. Wnt/LRP6 Signaling Functions in Transcriptionally-Silent Sperm. Immunostaining of phosphorylated LRP6 (p-LRP6; Tp1479) in mouse sperm. P-LRP6 marks activated Wnt/ LRP6 signaling, is largely restricted to the midpiece of sperm, and is superinduced by Wnt-containing exosomes released from the epididymis. DNA is shown in green.
[2_TD$IF]Wnt/STOP signaling functions in mature Xenopus oocytes to promote the first embryonic cell divisions [17]. Depleting Xenopus oocytes of maternal Lrp6 or Ccny, but not of b-catenin, leads to cell-cycle arrest at the one- or two-cell embryonic stage in a GSK3-dependent manner. This is because Wnt/STOP signaling in oocytes is necessary to stabilize mitotic effectors, including Plk1 and Ube2c. Likewise, Wnt/LRP6 signaling is required for maturation of transcriptionally-silent sperm [18]. Mice mutant for the Wnt/LRP6 signaling component Ccnyl1 are male-infertile and harbor immotile sperm [18,64,65]. Testicular sperm passing through the epididymis receive signals to terminally differentiate and to gain motility before ejaculation. The epididymal epithelium expresses [18_TD$IF]several [19_TD$IF]Wnts including Wnt2b, which is secreted in exosomes to the lumen. Wnt/ LRP6 signaling functions in sperm (Figure 2), where it elicits at least three b-catenin-independent responses via GSK3 repression [18,64]: first, it induces Wnt/STOP signaling to maintain protein homeostasis. Second, it establishes the membrane diffusion barrier in the sperm tail by promoting the clustering at the annulus of septin 4 (Sept4), a ring-forming protein controlling membrane dynamics. Finally, it initiates sperm motility by inhibiting PP1 phosphatase activity via reducing phosphorylation and binding of its inhibitory subunit PPP1R2. Consistently, overexpression of DKK1 in the epididymis reduces sperm motility, while truncated APC (APCmin[14_TD$IF]) increases it [18]. Importantly, the factors targeted by Wnt/LRP6 signaling in oocytes and sperm, including Sept4, PP1, and PLK1, play crucial roles in somatic cells as well, especially during mitosis. Thus, the lessons learned in germ cells may have broader relevance. Wnt/LRP6 Signaling in Neurons Neuronal connections are not only created de novo during development but are rewired in adult neural networks in an activity-dependent manner. The development and plasticity of synapses are often post-transcriptionally regulated in view of the long distances between synaptic terminals and the nucleus. Wnt/LRP6 signaling has emerged as a key regulator of axonal remodeling, synaptic plasticity, neurite outgrowth, and neurotransmitter release independently of b-catenin. In mouse cerebellum, Wnt7a localizes in synaptic areas, where it increases the size and spreading of the axonal growth cone [45,66]. Moreover, depletion of Wnt7a and Dvl1 impairs neurotransmitter release in mouse cerebellum [67–69]. Several lines of evidence suggest that these processes are modulated by Wnt/LRP6 signaling acting directly on the cytoskeleton. GSK3 phosphorylates the microtubule-associated proteins Tau, MAP1B, and MAP2, thereby destabilizing the microtubules at synaptic terminals [45,66]. Wnts reduce MAP1B phosphorylation, stabilize the microtubules, and induce axonal remodeling by inhibiting GSK3, independently of b-catenin or transcription [47,68]. Consistently, chemical inhibition of GSK3 mimics the Wnt7a remodeling effect in axons [47,66,68–70]. Likewise, Wnt/STOP signaling stabilizes Tau in non-neuronal cells [71].
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Tau phosphorylation and stability play crucial roles in axon guidance and maintenance, and its misregulation is a hallmark of some neurodegenerative disorders, notably Alzheimer [72,73]. In addition, LRP6 mutations are associated with late-onset Alzheimer's disease [74]. Thus, it appears promising to probe into the role of b-catenin-independent Wnt/LRP6 signaling in these disorders. Wnts also stimulate neurite outgrowth, and accumulating evidence suggests that this effect is mediated via Wnt/LRP6 signaling. First, chemical inhibition of GSK3 mimics Wnt-dependent neurite outgrowth, while Axin overexpression blocks it [47,66,75]. Second, APC localizes at the tip of hippocampal neurites with unphosphorylated Tau [76]. Third, although b-catenin plays structural roles in the neurite, its transcriptional activity is dispensable for neurite outgrowth [75,76]. Roles of b-catenin-independent Wnt/LRP6 signaling in neurons are evolutionarily conserved. In Drosophila, Wg (Wnt) inhibits Shaggy (GSK3) to stabilize synaptic microtubules through changes in the localization of Futsch (MAP1B) [77,78]. In addition, presynaptic Wg, Arrow (LRP6), and Shaggy, but not Armadillo (b-catenin), are required for neuromuscular junction development in Drosophila [78]. Similarly, Wg and Shaggy, but not Pangolin (TCF) or Armadillo, promote neuronal stability in the adult olfactory system [79]. In light of Wnt/STOP signaling, it appears fruitful to investigate which Shaggy/GSK3 target proteins modulate these effects in fly.
Crosstalk with other Signaling Cascades Cells typically receive multiple signals, and particular components act as signaling hubs that coordinate crosstalk between cascades. GSK3 is a key example of a signaling hub which not only integrates several inputs, such as Wnt, EGF, and insulin, but also multiple outputs (e.g., Table 1), thereby allowing Wnt/LRP6 signaling to directly regulate other pathways independently of b-catenin. During vertebrate axial patterning, the dorsal–ventral and anterior–posterior axes are regulated by BMP and Wnt gradients, respectively [80]. A key effector of the BMP pathway is Smad1, which is a Wnt/STOP target [38]. By stabilizing Smad1, Wnt/STOP signaling increases the duration of BMP signaling both in vitro and in Xenopus embryos, where it promotes the formation of the epidermis. Similarly, Wnt/STOP signaling regulates the stability of Smad4 [81,82], allowing Wnt ligands to enhance TGF-b signaling, but only in the presence of FGF. This is because GSK3dependent degradation of Smad4 requires priming phosphorylation by MAPK, which is activated by FGF. In the absence of FGF ligands, Wnt and TGF-b signaling remain insulated because GSK3 cannot phosphorylate the Smad4 degron [81]. Mutation of these sites inhibits germ-layer specification and Spemann–Mangold organizer formation during Xenopus development, highlighting the importance of this integrative network. Finally, Wnt/LRP6 signaling activates the Hippo pathway by stabilizing its transducer YAP/TAZ and inducing its nuclear translocation to promote differentiation of mesenchymal stem cells, among other processes [83,84]. Interestingly, YAP/TAZ accounts for a significant part of the Wnt transcriptional program, and is required in vivo for intestinal crypt overgrowth induced by APC deficiency [83].
Concluding Remarks and Future Directions The discovery of b-catenin-independent Wnt/LRP6 signaling branches raises the important question of the extent to which Wnt responses are mediated by transcriptional or b-cateninindependent routes (see Outstanding Questions). At the one extreme, during mouse gastrulation and Drosophila segmentation, Wnt3/Wg mutants phenocopy b-catenin/armadillo mutants, suggesting that transcriptional regulation is the dominant signaling mode [85–87]. At the other
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Outstanding Questions What are the post-transcriptional protein targets for Wnt/LRP6 signaling, notably in mitotic cells? Given the various other signaling inputs into GSK3 (e.g., insulin and EGF) and the large number of GSK3 substrates, what provides the specificity for different Wnt/LRP6 outputs? In which other biological processes does transcription-independent Wnt/ LRP6 signaling play a role? Does misregulation of b-catenin-independent Wnt/LRP6 signaling contribute to Wnt-associated diseases such as colorectal cancer?
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extreme, in transcriptionally-silent mouse sperm and frog eggs only b-catenin-independent Wnt signaling occurs. Even in HeLa cells, a 3 h Wnt3a pulse only induces the expression of three genes, a minor effect compared with the stabilization of 15% of the proteome through Wnt/ STOP, the modulation of the orientation of the mitotic spindle, and the upregulation of translation through the TOR pathway [42,43,46]. It is likely that other cell types will lie between these extremes and engage both b-catenin-dependent and -independent Wnt/LRP6 signaling branches. In dividing cells, a common theme among Wnt/LRP6-triggered signaling branches is that they all seem to orchestrate a growth program, for example by stimulating protein production via (i) increased gene expression (Wnt/b-catenin), (ii) enhanced protein biosynthesis (Wnt/TOR), and (iii) reduced protein degradation (Wnt/STOP). Another question is, given the various signaling inputs into GSK3 (e.g., insulin and EGF), what provides the specificity for the Wnt/LRP6 outputs? It has been proposed that only those GSK3 substrates that bind to the destruction complex, such as b-catenin and YAP/TAZ, can be targeted by Wnt ligands [88]. However, Wnt/LRP6 signaling depletes up to 70% of cytoplasmic GSK3 activity in multivesicular bodies, and this sequestration itself requires Axin [24,25,41]. Consistent with this, Wnt/LRP6 modulation of GSK3 targets other than b-catenin, such as YAP/ TAZ, Snail, or CNPY2, is also Axin/APC-dependent (Table 1). Thus, additional specificity may be provided by dedicated priming kinases for GSK3, as well as E3 ubiquitin ligases and their respective regulators. For instance, the TGFb effector Smad4 is only stabilized by Wnt/STOP signaling if gated by FGF, which activates MAPK and primes for GSK3, thus rendering Smad4 Wnt-sensitive [81]. Conversely, regulation of particular GSK3 targets, such as MAP1B, is shared between Wnt and EGF signaling [89]. Finally, the exploration of b-catenin-independent Wnt/LRP6 cascades may open novel options for the treatment of Wnt-associated diseases. Currently the pharmaceutical industry focuses on targeting the Wnt/b-catenin branch. However, as discussed here, misregulation of b-cateninindependent Wnt/LRP6 signaling affects cell growth and proliferation, as well as genome stability, which are hallmarks of cancer cells. Thus, there may be novel opportunities for pharmacological intervention with colorectal cancers harboring mutations upstream of GSK3 [28,90–94] by blocking b-catenin-independent Wnt/LRP6 branches. Acknowledgments We apologize to all authors whose primary work could not be cited owing to space constraints or whose work was inadvertently overlooked. This work was supported by the Deutsche Forschungsgemeinschaft.
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