Regulation by Wnt Signaling

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Regulation by Wnt Signaling Mrinmoy Pal Gene Regulation and Cellular Communication

Transcript of Regulation by Wnt Signaling

Page 1: Regulation by Wnt Signaling

Regulation by Wnt Signaling

Mrinmoy PalGene Regulation and Cellular Communication

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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.

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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".

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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.

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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.

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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

DkkNN

Canonical Pathway

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The Canonical Wnt PathwayThe 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 Grouchoco-repressor protein bound to the generegulatory protein LEF1/TCF. (B) Wntbinding to Frizzled and LRP clusters thetwo types of receptors together, resultingin phosphorylation of the cytosolic tailof LRP (by GSK3 and CK1) and activation of a cytoplasmic phosphoprotein named Dshevelled (Dsh). Axin binds to the phosphorylated LRP. The loss ofaxin from the degradation complexInactivates 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.

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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.

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The Canonical Wnt PathwayTarget Genes

• Members of the homeobox family: Transcription factorsEngrailed (en)Ultrabithorax (Ubx)• Genes expressed in development of the embryo: Required for organizer

formationSiamoisTwin• 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.

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The Canonical Wnt PathwayInduced 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.

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The Canonical Wnt PathwayInduced 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.

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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.

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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

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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.

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Wnt Signaling and Human Disease Gene DiseaseWnt3 Tetra-ameliaLRP5 Bone density defectsFz4 Familial Exudative Vitreoretinopathy (FEVR)

Axin2 Tooth agenesisPredisposition 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 limbsDecreased bone density: caused by loss of function mutation in LRPFEVR: 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

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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 humancolon, 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.

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