Canonical Wnt / β-Catenin Signaling

Background
Wnt Signaling Components
   a) Wnt protein  
   b) Receptors of Wnt proteins
   c) Wnt signaling modulators
Wnt /beta-catenin signaling mechanism
   a) Wnt signaling – Off
   b) Wnt signaling – On
   c) Wnt Target Genes
Wnt Signaling Functions
Conclusion

Background

Wnt signaling is critical in a myriad of biological functions, including cell fate determination, cell migration, cell polarity, organogenesis and neural patterning during embryogenesis¹. Wnt proteins in the stem cell niche control the behavior of various types of stem cells maintaining them in self-renewing state. The extracellular Wnt proteins stimulate several signal transduction cascades, which are classified into canonical or Wnt/β-catenin pathway and non-canonical or β-catenin-independent pathway². Wnt/β-catenin signaling is well studied and the current review focuses on components and signaling mechanism of β-catenin dependent signaling.

Wnt Signaling Components

a) Wnt protein

Wnt is ubiquitously expressed in all metazoan animals. There are 19 mammalian genes that code for cysteine rich Wnt proteins, falling into 12 conserved Wnt subfamilies.  Wnt proteins are globular, approximately 40 KDa; the amino-terminal domain majorly consists of α-helices with five disulfide bridges while the carboxy-terminal domain is dominated by two β-sheets with six disulfide bonds. Wnt proteins undergo post-translational modifications before they are released into the extracellular cytoplasmic milieu. Wnt is glycosylated and palmitoylated by endoplasmic reticulum protein called Porcupine³ (Porc) and transported to the plasma membrane by Wntless proteins (Wls). Loss of either Porc⁴ or Wls⁵ prevents the secretion of Wnt proteins, leading to birth defects.  Wnt proteins are known to act in contact dependent manner as well as across distant tissues. Wnt/β-catenin pathway is the result of a prominent close-range signaling along with beta-catenin⁶.

b) Receptors of Wnt proteins

Wnt lipoprotein binds to a receptor complex that comprises of Frizzled (Fz) and lipopolysacharide like receptor protein 5/6 (LRP5/6). The frizzled protein has seven-transmembrane domains and a large extracellular N-terminal cysteine rich domain that provides platform for Wnt binding. The interaction between Wnt and Fz protein is not specific; a single Wnt protein can bind to multiple Fz proteins. Unlike Fz protein, LRP6 has specific binding sites for different classes of Wnt proteins⁷.  Ligand (Wnt) binding brings conformational changes within the receptors (LRP6), activating kinases like GSK3 and CK1γ⁸. Both GSK3⁹ and CK1γ¹⁰ eventually phosphorylate several signaling components of Wnt pathway, including β-catenin, Axin, APC and LRPs. Little is known about the role of Fz in Wnt pathway. Upon the signal reception, the cytoplasmic domain of Fz receptor interacts with Dishevelled (Dsh) proteins and facilitates the interaction between LRP tail and Axin. Both Axin and Dsh proteins bind each other through DIX domain and mediate the formation of LRP-Fz dimers.

c) Wnt signaling modulators

The Wnt/β-catenin signaling is modulated by extracellular ligands listed in the table below.

Table 1 Modulators of Wnt/β-catenin signals

Modulator	Effects
Frizzled-related proteins (sFRPs)11	Inhibition of Wnt
Wnt inhibitory protein11	Inhibition of Wnt
Dickkopf (DKK)12	Inhibition of LRP5/6
WISE/SOST family proteins12	Inhibition of LRP5/6
APCDD113	Inhibition of Wnt and LRP
Norrin14	Activation of Fz/LRP complex
R-spondins15	Activation of Fz/LRP complex, Lgr receptors

Wnt /beta-catenin signaling mechanism

The stability of cytoplasmic β-catenin is critical for the signaling output of Wnt signaling. β-catenin (781 amino acids) consists of central region of 141-664 amino acids made up of armadillo repeats, flanked by distinct N- and C-terminal domains (NTD and CTD). Although, the NTD and CTD are structurally flexible, the central region is rigid and serves as interaction platform for binding proteins in cytosol and in the nucleus. The stability of β-catenin depends on what is now termed as the “cytoplasmic destruction complex” comprising Axin, two tumor suppressor proteins APC and WTX and two constitutively active kinases, CK1α/δ and GSK3α/β.

a) Wnt signaling - ‘Off’

In the absence of Wnt, the fate of β-catenin is determined by kinases, CK1 and GSK3.

• Phosphorylation: β-catenin is phosphorylated by CK1 at Ser45, Ser33 and Ser37, and by GSK3 at Thr41
• Ubiquitination: Phosphorylated β-catenin is targeted for β-Trcp-mediated ubiquitination and subsequent degradation by proteasome¹⁶.
• Transcriptional repression: In the absence of β-catenin, transcriptional factors like TCF engage with Groucho, a transcriptional repressor which prevents the transcription of target genes.

b) Wnt signaling - ‘On’

In the presence of Wnt ligands a signaling cascade is initiated.

• Disassociation of destruction complex: Binding of Wnt ligands to the Frizzled receptors and LRP5/6, triggers a series of events that ultimately disrupts the APC/Axin/GSK3β (destruction) complex and stabilizes the β-catenin.
• Translocation of β-catenin: β-catenin accumulates in the cytoplasm and is free to translocate into the nucleus.
• Transcriptional regulation: Within the nucleus, β-catenin acts as transcriptional coactivator of transcriptional factors of the TCF/LEF family. Other binding partners Legless and Pygopus maintain the nuclear retention and transactivation ability of β-catenin^17–19.

Figure 1. Wnt /β-Catenin signaling pathway

c) Wnt target genes

The genes regulated by Wnt signaling include those of transcription factors, ECM components, cell adhesion proteins, enzymes and hormones (Table 2).

Table 2 Target genes of β-catenin and their modulation

Modulation	Genes
Upregulation	C-myc20, Tcf-121, LEF-122, PPAR-delta23, c-jun24, MMP-725, Axin-226, Nr-CAM27, Claudin-128, VEGF29
Down regulation	Osteocalcin30, E-cadherin31

Wnt Signaling Functions

Among the wide spread effects of Wnt signaling on target cells, the role in regulating the plasticity of stem cells drawn particular interest.

Embryonic stem cells: Wnt canonical pathway maintains self-renewal of embryonic stem cells.  Wnt agonist like R-Spondin mediates pluripotency in mouse embryonic stem cells, which have clinical implication in the treatment of degenerative diseases^32,33.

Mesenchymal stem cells: The activation of canonical Wnt pathway promotes osteogeneic differentiation of mesenchymal stem cells. Signal intensity of Wnt/β-catenin signal determine the fate of mesenchymal stem cells, where proliferation and self-renewal were induced under low levels of Wnt/β-catenin, while osteogenic differentiation is triggered under high levels of Wnt signaling³⁴.

Intestine stem cells: Wnt/β-catenin regulates stem cell differentiation in intestine. Inhibition of Wnt signaling mediated by the expression of Dkk-1 induces complete loss of crypts, source of self-renewing tissues in intestine. In contrast, activation of Wnt signaling stimulates the proliferation of crypt progenitors.

Hematopoietic Stem Cells: Wnt/β-catenin activation increases the hematopoietic progenitors. Knockdown of Wnt3a decreased the progenitor cells³⁵ and overexpression of activated β-catenin triggered the expansion of hematopoietic stem cells³⁶.

Hair follicle stem cells: Wnt signaling is important for establishment of hair follicle and activates stem cells. Conditional loss of β-catenin in skin epithelia leads to hair follicle stem cells depletion³⁷.

Regulation of microtubules: Wnt regulates the stability and organization of microtubules that influence alignment of mitotic spindles and segregation of chromosomes during cell division; these in turn influence cell migration and polarization. Wnt also induce changes in morphology and behavior of axons via tethering of microtubules, which decrease the axon extension and increase the axon branching³⁸.

Cancer metabolism: Canonical Wnt pathway and downstream effectors regulates cell proliferation, cell death, senescence and metastasis³⁹. Components of Wnt pathway are frequently mutated (Table 2), which implicates its role cancer progression.

Table 3 Mutations of Wnt/β-catenin components associated with cancer

Mutation	Protein	Disease
Gain of function	β-catenin	Colon cancer40
	TC4	Colon cancer41
	LRP5	Hyperparathyroid tumors42
Loss of function	LEF1	Sebaceous skin tumor43
	AXIN1	Hepatocellular cancer44
	AXIN2	Colorectal cancer45
	APC	Colorectal cancer46

Conclusion

Wnt signaling is conserved throughout the evolution and there is a strong correlation between deregulated Wnt signals and multiple disease conditions. Wnt proteins are active in stem cells of neural, mammary and embryonic tissues. It has been reported that while defined factors such as LIF, Basic FGF, Hedgehog, BMP-4 maintain embryonic stem cells in undifferentiated state, Wnt proteins play a role in ES cell control. Interplay of Wnt proteins and other factors including small molecules like retinoic acid for maintenance, self-renewal and differentiation of stem cells is a prominent area of research that has vast implications in the way stem cells are shaping up the current disease therapy options.

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