The Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signaling has also been found to be associated with cancer. Understanding the regulation by Wnt signaling may serve as a paradigm for understanding the dual nature of self-renewal signals.

1. Regulation by Wnt
Signaling
Mrinmoy Pal
Gene Regulation and Cellular
Communication

2. Introduction
• The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that
regulates crucial aspects of cell fate determination, cell migration, cell polarity, and
organogenesis during embryonic development.
• Till date, Three major Wnt signaling pathways have been characterized:
(a) The canonical Wnt pathway: leads to regulation of gene transcription.
(b) The non-canonical planar cell polarity (PCP) pathway: regulates
the cytoskeleton.
(c) The non-canonical Wnt/calcium pathway: modulates levels
of calcium inside the cell.
• Defective Wnt signaling is a causative factor for a number of pleiotropic human
pathologies. Most notably, these pathologies include cancers of the breast, colon
and skin, skeletal defects and human birth defect disorders.

3. History and Etymology
• 1982: Roel Nusse and Harold Varmus infected mice with mouse mammary tumor
virus (MMTV) in order to mutate mouse genes to see which mutated genes could
cause breast tumors. They identified a new mouse proto-oncogene that they named
int1 (integration1).
• 1987 : Scientists realized that the int1 gene in Drosophila was already known and
characterized as Wingless or Wg. This segment polarity gene was found to be
involved in body axis pattering during embryonic development. Taking cue from
this, researchers determined that the mammalian int1 (discovered in mice) is also
involved development.
• Continued research led to the discovery of further int1-related genes; however,
beause those genes were not identified in the same manner as int1, the int-gene
nomenclature was inadequate. Thus, the int/Wingless family became the Wnt
family. The name Wnt is a portmanteau of ‘Wg’ and ‘int’, which stands for
"Wingless-related integration site".

4. Wnt Proteins
• Wnt comprises a diverse family of secreted lipid-modified signaling glycoproteins that
are 350–400 amino acids in length.
• A signal sequence of 15 to 30 amino acids occurrs at the N-terminal of the precursors
of these secretory proteins; it is required for transport of the protein across the
membrane of the RER into the cisternae, where it is immediately cleaved off by an
endopeptidase.
• The type of lipid modification that occurs on these proteins is palmitoylation
of cysteines in a conserved pattern of 23–24 cysteine residues. Presence of this lipid
moiety targets Wnt to the membrane. Mutation of cysteine or removal of palmitate
inactivates Wnt.
• Wnt proteins also undergo glycosylation, which insures proper folding and secretion.
• In Wnt signaling, these proteins act as ligands to activate the different Wnt pathways
via paracrine and autocrine routes. These proteins are highly conserved across species.

5. Mechanism: Foundation
Wnt signaling begins when a Wnt protein binds to the N-terminal extra-cellular cysteine-rich domain of a Frizzled (Fz)
family receptor. These receptors span the plasma membrane seven times just like the GPCRs. However, to facilitate Wnt
signaling, co-receptors may be required alongside the interaction between the Wnt protein and Fz receptor. Examples
include lipoprotein receptor-related protein (LRP)-5/6 & receptor tyrosine kinase (RTK). Upon activation of the receptor, a
signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. This signal is transmitted via a
direct interaction between Fz and Dsh. Dsh proteins are present in all organisms and they all share the following highly
conserved protein domains: an amino-terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain.
These different domains are important because after Dsh, the Wnt signal can branch off into multiple pathways and each
pathway interacts with a different combination of the three domains.

6. Extra-Cellular Regulators
• Extracellular enhancer: Binds and stabilizes Wnt
proteins which further limits diffusion and
modulates their signaling abilities.
– HSPG -Heparin-sulfated forms of
proteoglycans
• Extracellular inhibitors: Prevents interaction of Wnt
proteins with Fz to antagonize Wnt signaling.
– SFRP (Secreted Frizzled-related protein), WIF
(Wnt inhibitory factor) - Resembles ligand-
binding domain of Frizzled
• Non -Wnt proteins that interact with Wnt receptors:
– Dickkopf (Dkk)- Binds with co-receptor LRP6
and another transmembrane protein Kremen,
gets endocytosed and depletes LRP6 from
membrane.
– Norrin- No sequence similarity to Wnt, still
the ligand binds to Fz and induces canonical
signalling pathway
SFRP
WIF
DkkN
N
Canonical
Pathway

7. The Canonical Wnt Pathway
The Wnt/β-catenin signaling pathway.
(A) In the absence of a Wnt signal,
cytoplasmic β-catenin that is not bound to
cadherin proteins is degraded by a destruction
complex containing APC, axin, GSK3, and
CK1. In this complex, β-catenin is
phosphorylated by CK1 and then by GSK3,
triggering its ubiquitylation and
degradation in proteasomes. Wnt-responsive
genes are kept inactive by the Groucho
co-repressor protein bound to the gene
regulatory protein LEF1/TCF. (B) Wnt
binding to Frizzled and LRP clusters the
two types of receptors together, resulting
in phosphorylation of the cytosolic tail
of LRP (by GSK3 and CK1) and activation
of a cytoplasmic phosphoprotein named
Dshevelled (Dsh). Axin binds to the
phosphorylated LRP. The loss of
axin from the degradation complex
Inactivates it. Moreover, DIX and PDZ
domains of the activated-Dshevelled protein
inhibit the GSK3 activity. This allows
unphosphorylated β-catenin to accumulate
and translocate to the nucleus. Once in In the
nucleus, β-catenin binds to TCF/LEF family
of transcription factors, displaces the co-
repressor Groucho, and acts as a coactivator
to stimulate the transcription of Wnt target
genes.

8. The Canonical Wnt Pathway
Nuclear Activity of β-Catenin. TCF provides sequence-specific binding activity and, in the absence of nuclear β-catenin,
partners with the transcriptional repressor Groucho and histone deacetylases to form a repressive complex. When β-catenin enters
the nucleus, it directly replaces Groucho and converts the complex to a transcriptional activator, thereby effecting the
transcription of Wnt target genes. Other members of this activating complex are the histone acetylase CBP/p300, and the
SWI/SNF complex member Brg-1. Lgs and Pygo also bind to β-catenin, possibly driving its nuclear localization. Negative
regulation of signaling is provided by NLK (Nemo-like kinase: which phosphorylates TCF, sending it to the cytoplasm], and
ICAT (inhibitor of catenin: disassociates TCF/ β-catenin-CBP complex) and Chibby, which are antagonists of β-catenin. In
addition to TCF, two other DNA-binding proteins have been shown to associate with β-catenin: Pitx2 and Prop1. In the case of
Prop1, β-catenin can act as a transcriptional activator or repressor of specific genes, depending on the co-factors present.
However, the participation of any particular β-catenin complex in transcriptional regulation is highly cell type-dependent.

9. The Canonical Wnt Pathway
Target Genes
•Members of the homeobox family: Transcription factors
Engrailed (en)
Ultrabithorax (Ubx)
•Genes expressed in development of the embryo: Required for organizer formation
Siamois
Twin
•Cellular proliferation genes: Control the G1 to S phase transition in the cell cycle
c-Myc
Cyclin D1
•Wnt signaling components: Feedback control of canonical Wnt pathway
Fz
Dfz2
Arrow/LRP
HSPG
Nemo
Dfz2 is downregulated by Wg. This reduces the levels of a high-affinity receptor
that might otherwise limit Wg distribution and allows Wg to diffuse over longer
distances.

10. The Canonical Wnt Pathway
Induced Cell Response
• Axis patterning in Xenopus embryo:
Fertilization, through a reorganization of the microtubule cytoskeleton, triggers a 30°
rotation of the egg cortex, relative to the core of the egg, in a direction determined by
the site of sperm entry. The resulting dorsal vegetal concentration of maternal Wnt11
mRNA leads to the production of the Wnt11 signal protein and forms the Spemann-
Mangold organizer which establishes the dorsoventral polarity in the future embryo.

11. The Canonical Wnt Pathway
Induced Cell Response
Cell Proliferation and Segregation in gut
•
Wnt signaling maintains proliferation in
the crypt, where the intestinal stem cells
reside. It also drives expression of the
components of the Notch signaling
pathway in that region; Notch signaling
through lateral inhibition, forces cells
there to diversify.
Cells in the crypt express EphB
proteins, while the differentiated
cells that cover the villi express
ephrinB. The repulsive cell–cell
interaction mediated by encounters
between these two types of
cell-surface molecules keeps the
two classes of cells segregated.

12. Non-canonical Planar Cell Polarity Pathway
•
The noncanonical planar cell polarity (PCP) pathway does
not use LRP-5/6 as its co-receptor and is thought to
use receptor tyrosine kinases like PTK7 or ROR2. The PCP
pathway is activated via the binding of Wnt to Fz and its
co-receptor. The receptor then recruits Dsh, which uses its
PDZ and DIX domains to form a complex with
Dishevelled-associated activator of morphogenesis 1
(DAAM1). Daam1 then activates the small GTPase Rho
through a guanine exchange factor (WGEF).
Rho activates Rho-associated kinase (ROCK), which leads
to modification of actin cytoskeleton. Parallely, the C-
terminal DEP domain of Dsh activates Rac GTPase and
mediates profilin binding to actin. Rac can also
activate JNK and lead to actin polymerization and
cytoskeletal modulation.
•The PCP pathway emerged from genetic studies in Drosophila
•Mutations in Wnt signaling components were found to randomize the orientation of epithelial structures.
•The defining feature of this pathway is its regulation of the actin cytoskeleton for such polarized
organization of structures and directed migration.

13. Induced Cell Response
• Asymmetric division during C. elegans embryogenesis:
A Wnt signal from the P2 precursorcell causes the EMS cell to orient its mitotic spindle and generate two founder
daughters that become committed to different fates as a result of their different exposure to Wnt protein—the MS cell
and the E cell (the founder cell of the gut).
•Divison of sensory mother cell during bristle development in Drosophila:
The planar polarity in the initial division of the sensory mother cell is controlled by a PCP pathway. This planar cell
polarity is basically associated with asymmetric localization of the receptor Frizzled itself to one side of the cell.
•Regulates Convergent Extension during Xenopus gastrulation.
Polarized cells intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and
anteroposterior elongation (extension)
Non-canonical Planar Cell Polarity Pathway

14. Non-canonical Wnt/Ca Pathway
+2
A schematic representation of the Wnt/Ca2+ signal
transduction cascade. Wnt signaling via Fz mediates
activation of Dsh via activation of G-proteins. Dishevelled
activates the cGMP-specific phosphodiesterase (PDE) which
inhibits PKG and in turn inhibits Ca2+ release. Dsh through
PLC activates IP3, which leads to release of intracellular
Ca2+, which in turn activates calcium/calmodulin-dependent
kinase II (CamKII) and calcineurin. Calcineurin activate NF-
AT to regulate ventral cell fates. CamK11 activates TAK1
and NLK, which inhibit β-catenin/TCF function to negatively
regulate dorsal axis formation. DAG through PKC activates
CDC42 to mediate tissue separation and convergent
extension (CE) movements during gastrulation.
•The noncanonical Wnt/calcium pathway also does not involve β-catenin.
•Its role is to help regulate calcium release from the ER in order to control intracellular calcium levels.
•Wnt/Ca2+ pathway functions as a critical modulator of both the canonical and PCP pathways.

15. Wnt Signaling and Human Disease
Gene Disease
Wnt3 Tetra-amelia
LRP5 Bone density defects
Fz4 Familial Exudative Vitreoretinopathy (FEVR)
Axin2 Tooth agenesis
Predisposition to Colorectal Cancer
APC Familial adenomatous polyposis (FAP)
Colon Cancer
Extracellular Wnt Protein Target Cell Membrane Protein Intracellular Protein
Tetra-amelia: Loss of function Wnt 3 mutation, rare human genetic disorder, absence of all four limbs
Decreased bone density: caused by loss of function mutation in LRP
FEVR: Fz4 mutated in seventh transmembrane domain leading to loss of Fz4/LRP signaling causes
progressive vision loss. The disorder prevents blood vessels from forming at the edges of the retina, which
reduces the blood supply to this tissue.
Oligodontia: Nonsense mutation in Axin2 leading to a condition where multiple permanent teeth are
missing

16. Mutations inhibit APC’s ability to bind β-catenin; thus, β -catenin accumulates in the nucleus and stimulates
the transcription of c-Myc and other Wnt target genes, even in the absence of Wnt signaling. The resulting
uncontrolled cell growth promotes the development of adenoma and colon cancer.
Wnt Signaling and Human Disease
Familial adenomatous polyposis (FAP) & Colon Cancer
An adenoma in the human
colon, compared with normal
tissue from an adjacent region
of the same person’s colon. The
specimen is from a patient with an
inherited mutation in one of his
two copies of the Apc gene. A
mutation in the other Apc gene
copy, occurring in a colon
epithelial cell during adult life,
has given rise to a clone of cells
that behave as though the Wnt
signaling pathway is permanently
activated.

17. References
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signalling".Current Opinion in Structural Biology 29: 77–84.