v468.chapter 1 经典 Wnt/b-Catenin 信号通路 （英文完整版）

Methods in Molecular Biology v468. chapter 1

Abstract

Embryonic development of multicellular organisms is an incredibly complex process that relies heavilyon evolutionarily conserved signalling pathways to provide crucial cell–cell communication. Typically,secreted signalling proteins such as Wnts, BMPs, and Hedgehogs released by one cell population willtrigger concentration-dependent responses in other cells located some distance away. In adults, the samesignalling pathways orchestrate tissue renewal in organs such as the intestine and skin, and direct tissueregeneration in many organs following injury. Strict regulation of these signalling pathways is critical,with insufficient or excess activity having catastrophic consequences including severe developmentaldefects or, later in life, cancer. This chapter deals with the b-catenin-dependent branch of Wnt signalling(also referred to the canonical pathway).

Key words: Wnt , Morphogen , b-catenin , Groucho , Tcf , Target gene , Constitutive activation , Stem cell ,Colon cancer.



1. Introduction

The Wnt pathway derives its name from the Drosophila (fruit-fly)W ingless gene and the mouse I NT -1 gene. The mouse Wnt1gene (originally named Int1), was identified in 1982 as a geneinappropriately activated by integration of the Mouse MammaryTumor Virus in virally induced breast tumors (1) . This establishedthe first link between mis-expression of Wnt genes andcancer (branding Wnt-1 a proto-oncogene) and revealed Wnt-1to be a secreted cysteine-rich protein with the potential to act asa signalling molecule. However, the real breakthrough in linking thisgene to a signalling pathway was made by fly geneticists, who demonstrated that the Drosophila Wnt1 counterpart, Wingless(Wg) was a crucial component of a novel signal transductionpathway controlling body patterning during larval development(2) . This pathway was subsequently found to be highly conservedin frogs, where it orchestrates proper induction of the body axisduring embryonic development. A particularly graphic exampleof the influence of the Wnt pathway on vertebrate developmentwas provided by McMahon and Moon in 1989, when they demonstratedthat forced activation of the Wnt pathway at the ventral(anterior) side of early frog embryos by injection of Wnt1messenger RNA (mRNA) caused a complete duplication of thebody axis and, as a consequence, the development of two-headedembryos (3) . More recently, altered Wnt signalling activity inadults has been linked to a wide range of human diseases, includingcancer, bone defects, schizophrenia, and arthritis (4 , 5) .

This chapter aims to provide a simplified overview of how Wntproteins are produced and secreted and how they subsequently activatethe canonical Wnt signalling pathway in recipient cells to effectchanges in cell growth, movement, and cell survival.

2. The Wnt Family

Since the identification of Wnt1, genome sequencing hasrevealed the existence of another 18 Wnt genes in mammals,which can be divided into 12 highly conserved subfamilies onthe basis of sequence similarity (6) . Many of these subfamiliesare highly conserved in early multicellular organisms such as thesea anemone, highlighting the crucial role of the Wnt pathwayin driving body patterning throughout the animal kingdom. AllWnt proteins share common features that are essential for theirfunction, including a signal peptide for secretion, many potentialglycosylation sites and multiple cysteine residues responsiblefor ensuring proper folding and secretion. With a few exceptions(Wg, Wnt3/5, and Wnt4), Wnt proteins are generally around350 amino acids long and have an approximate molecular weightof 40 kDa (7) .

3. Wnt Secretion and Delivery

The recent success in developing cell-based systems for expressingand purifying biologically active Wnt proteins has providedinvaluable insights into how Wnts are converted from immature precursors in the cell into secreted signalling molecules capableof interacting with specific receptor complexes on target cellslocated up to 20–30 cell diameters distant (8 , 9) .

Wnts start out as precursors containing an N-terminalhydrophobic signal peptide that directs the immature proteinto the endoplasmic reticulum (ER). In the ER, the signal peptideis cleaved off by a resident protease and the Wnt proteinis extensively modified by the addition of sugars and lipids toensure efficient secretion, intercellular delivery, and to maximizebiological activity on target cells. Probably the most significantmodification to occur is the attachment of a palmitate moietyto a conserved cysteine residue on the Wnts, thereby convertingthem into hydrophobic proteins. This modification was shown tobe essential for the biological activity of Wnt proteins producedin cell lines, when inhibition of essential acyltransferase enzymeactivity (typically required for lipid modifications) or mutation ofthe conserved Wnt cysteine modification site resulted in a proteinthat was neither hydrophobic nor active (8 , 10) . It has beenproposed that this lipid modification serves to anchor the Wntproteins in the vicinity of the oligosaccharyl complex (OST) atthe ER membrane, thereby facilitating their efficient N -linkedglycosylation at conserved asparagine residues (11) . Alternatively,the palmitate moiety may prevent the modified cysteine residuefrom forming disulphide bonds that would otherwise result inmis-folding and retention of the Wnt protein in the ER. TheER-resident acyltransferase enzyme believed to perform this lipidmodification in vivo is encoded by the Drosophila porcupine geneand its homologues ( mom-1 in worms) (9 , 10 , 12) .

Fig. 1.1. Wnt secretion. To facilitate their secretion, Wnt proteins must first be palmitoylatedin the endoplasmic reticulum (ER) by the actions of Porcupine. Wnt proteinsmust also complex with Wntless (Wls/Evi) in the Golgi to be efficiently routed to theoutside of the cell. Loading onto lipoprotein particles may occur in a dedicated endocytic/exocytic compartment. The retromer complex may shuttle Wls between the Golgi andthe endo/exocytic compartment. Reprinted with permission from ref. (4) . CopyrightElsevier (2006).

More recently, it has become increasingly clear that efficientsecretion of Wnt proteins is a complex process requiring the concertedactions of several conserved genes (Fig. 1.1 ). This is highlightedby the discovery of another gene termed Wntless / evenessinterrupted , which, like Porcupine , is also indispensable for Wntsecretion in Drosophila (13 , 14) . Wntless encodes a seven-passtransmembrane protein that is highly conserved across speciesfrom worms (mom-3) to man (hWLS). Inactivation of this genein Wnt-producing cells short-circuits the Wnt secretion processand leads to retention of the Wnt protein inside the cell. Althoughthe precise function of Wntless remains elusive, it largely residesin the Golgi apparatus, where it physically interacts with the Wntproteins. This has prompted speculation that it may act as a chaperoneto guide the Wnt proteins through other post-translationalmodifications necessary for their efficient secretion and/or regulatethe intracellular trafficking of Wnt between different cellcompartments en route to its release into the extracellular space.

Genetic screens in Caenorhabditis elegans have revealed yetmore proteins involved in controlling the fate of Wnt as Wntpasses through the cell secretion machinery. These proteins reside in a complex called the retromer, which is involved in intracellulartrafficking in many species ranging from yeast to man (15) .

Unlike Porcupine and Wntless mutations, abrogation of retromerfunction in mammalian Wnt-producing cell lines does not substantiallyimpair Wnt secretion. Instead, depletion of the retromercomplex in worms and frogs appears to prevent long-rangetransport of secreted Wnts to their target cells. In contrast, deliveryof the Wnt proteins to neighboring target cells (short-rangesignalling) remains largely intact. These observations have led tothe proposal that retromers direct Wnt proteins from the Golgiapparatus into specialized intracellular compartments dedicatedto long-range Wnt secretion.

Wnts are classic morphogens (long-range signalling moleculeswhose activity is concentration dependent) and must therefore form long-range concentration gradients capable ofactivating signalling on target cells up to 20–30 cell diametersdistant from their source (16) . It is likely that the various posttranslationalmodifications bestowed on Wnt whilst en routethrough the cell play an important part in setting up these concentrationgradients. For instance, the addition of palmitate tothe Wnts may facilitate the association of Wnts with lipoproteinparticles involved in lipid transport (referred to as argosomes)(17) . This interaction may dislodge Wnts from cell membranes inthe immediate vicinity of the Wnt source thereby allowing themto spread further afield. Alternatively, the argosomes may simplycapture and concentrate the Wnt proteins to a level required forefficient activation of the pathway on distant target cells. Geneticstudies in Drosophila indicate that interaction with heparin sulphateproteoglycans (HSPG) present on cell membranes and theextracellular matrix is also likely to play a role in transportingand stabilizing Wnts (6 , 18) . Finally, the Wnts may be activelytransported by cytonemes, which are long, thin, filopodial cellprocesses present in the extracellular matrix (19) .

Binding of these secreted Wnt proteins to specific receptorcomplexes on target cells activates one of three intracellularsignalling pathways: the canonical (T-cell factor [Tcf]/β-catenin)pathway, the non-canonical (planar cell polarity) pathway and theWnt/Ca 2+ pathway (20 , 21) . Each of these pathways delivers avery different set of instructions to the recipient cell by activatingspecific sets of target genes. This chapter focuses on the canonicalpathway, which is better characterized and generally consideredto be more relevant for cancer development.

4. The Canonical Wnt Pathway

The canonical Wnt pathway strictly controls the levels of a cytoplasmicprotein known as β-catenin, which has crucial roles inboth cell adhesion and activation of Wnt target genes in thenucleus (4) . In the absence of a Wnt signal, β-catenin is efficientlycaptured by a scaffold protein termed Axin, which ispresent within a protein complex (referred to as the destructioncomplex) that also harbors adenomatous polyposis coli (APC)and the protein kinases casein kinase (CK)-1 and glycogen synthasekinase (GSK)-3 (Fig. 1.2 , left panel). APC is an essentialcomponent of the destruction complex, where it is thought toensure the efficient recruitment and anchoring of β-catenin. Theresident CK1 and GSK3 protein kinases sequentially phosphorylateconserved serine and threonine residues in the N-terminusof the trapped β-catenin (22) , generating a binding site for an E3 ubiquitin ligase, which subsequently targets the β-cateninfor rapid proteasomal degradation (23) . Such efficient suppressionof β-catenin levels ensures that Groucho proteins are free tobind Tcf/lymphoid enhancer factor (Lef) proteins occupying the promoters and enhancers of Wnt target genes in the nucleus(24 , 25) . These Tcf/Groucho complexes actively suppress the transcriptionalactivation of Wnt target genes such as c-Myc, therebysilencing an array of biological responses, including cell proliferation.

Fig. 1.2. Overview of the canonical Wnt signalling pathway. A Wnt off : in the absence of a Wnt signal, b-catenin is capturedby APC and Axin within the destruction complex, facilitating its phosphorylation ( P ) by the kinases CK1a and GSK3.CK1a and GSK3 then sequentially phosphorylate a conserved set of serine and threonine residues at the N-terminus ofb-catenin. This facilitates binding of the b-transducin repeat-containing protein (b-TRCP), which subsequently mediatesthe ubiquitylation ( Ub ) and efficient proteasomal degradation of b-catenin. The resulting b-catenin “drought” ensuresthat nuclear DNA-binding proteins of the Tcf/Lef transcription factor family (Tcf-1, Tcf-3, Tcf-4, and Lef-1) actively represstarget genes by recruiting transcriptional co-repressors (Groucho/TLE) to their promoters and/or enhancers. B Wnt on : interaction of a Wnt ligand with its specific receptor complex containing a Frizzled family member and low-density lipidreceptor (LRP)-5 or LRP6 triggers the formation of Dishevelled (Dvl)/Frizzled (Fzd) complexes. The resulting generationof LRP/Fzd/Dsh aggregates at the cell membrane induces the phosphorylation of LRP by CK1a, thereby facilitating relocationof Axin to the membrane and inactivation of the destruction box. This allows b-catenin to accumulate and enterthe nucleus, where it interacts with members of the Tcf/Lef family. In the nucleus, b-catenin converts the Tcf proteinsinto potent transcriptional activators by displacing Groucho/TLE proteins and recruiting an array of coactivator proteinsincluding CBP, TBP, BRG-1, Bcl9, Legless, Mediator, and Hyrax. This ensures efficient activation of Tcf target genes suchas c-Myc, which instruct the cell to actively proliferate and remain in an undifferentiated state. Following dissipation ofthe Wnt signal, b-catenin is evicted from the nucleus by the APC protein, and Tcf proteins revert to actively repressingthe target gene program. APC , adenomatous polyposis coli; CK1 a, casein kinase 1a; CBP , CREB-binding protein;Tcf , T-cell factor; Lef , lymphoid enhancer factor; Bcl9 , B-cell lymphoma-9; Dvl, ; b-cat, b-catenin; PYG , Pygopus. (For more details, I recommend the Wnt homepage: http://www.stanford.edu/~rnusse/Wntwindow.html ). Reprinted withpermission from ref. (5) . Copyright Nature Publishing Group (2006).

Rapid activation of the canonical pathway occurs when Wntproteins interact with specific cell surface receptor complexescomprising members of the Frizzled family of seven-pass transmembraneproteins and the single-pass transmembrane proteins,low-density lipid receptor (LRP)-5 or LRP6 (Fig. 1.2 , rightpanel). This triggers the phosphorylation of Dsh proteins andpromotes their interaction with the Frizzled proteins (26) . Theresulting Dsh/receptor complexes are thought to stimulate theformation of LRP6 aggregates at the membrane, which facilitatesthe phosphorylation of the LRP6 intracellular tails by the CK1γ.

As a consequence, Axin is recruited to this receptor complex andthe proteasomal degradation of β-catenin is blocked (22 , 27 , 28) .

This allows β-catenin to accumulate and enter the nucleus, whereit interacts with members of the Tcf/Lef family and converts theminto potent transcriptional activators by recruiting co-activatorproteins and ensuring efficient activation of Wnt target genes.

5. Regulation of Wnt Target Gene Activity

In the absence of Wnt signalling, Tcf proteins occupy target geneenhancers and promoters independently of β-catenin. There arefour family members in vertebrates, Tcf-1, Tcf-3, Tcf-4, and Lef-1,which share a highly similar DNA-binding domain termed thehigh mobility group (HMG) box. This HMG box provides thetarget gene specificity of the Tcf proteins by ensuring that theyexclusively bind DNA at a conserved motif defined as AGA/TA/TCAAAG (29 , 30) . Interaction of Tcf proteins with this motif inenhancers and promoters of target genes causes the DNA to dramaticallybend through more than 90 degrees. When β-catenin isabsent from the nucleus, the Tcf proteins bound to the Wnt targetgenes act as transcriptional repressors by recruiting membersof the Groucho/TLE protein family (24 , 25) .

Relocation of β-catenin from the cytoplasm to the nucleusfollowing its Wnt-induced stabilization is essential for achievingthe efficient activation of Wnt target genes and ensuring theappropriate physiological response. Exactly how this is achievedremains somewhat of a mystery. Its nuclear import appears independentof the nuclear localization signal (NLS)/importin machinery;although β-catenin is itself related to the importin/karyophilinprotein family and may gain access through direct interaction withthe nuclear pores. There has also been speculation that β-catenin may shuttle to the nucleus as a complex with other proteins suchas Pygopus/B-cell lymphoma (Bcl)-9 and the Tcf/Lef family,which are actively imported by virtue of their NLS. However,this is unlikely to be the major access route because β-catenin stilllocalizes to the nucleus in the absence of these proteins (31) .

Fig. 1.3. Transactivation of Wnt target genes. The Tcf/b-catenin complex interacts witha variety of chromatin-remodelling complexes to activate transcription of Wnt targetgenes. The recruitment of b-catenin to Tcf target genes affects local chromatin in severalways. B-cell lymphoma (Bcl) 9 acts as a bridge between Pygopus and the N-terminusof b-catenin. Evidence suggests that this trimeric complex is involved in nuclearimport/retention of b-catenin, but may also directly enhance the ability of b-catenin toactivate transcription. The C-terminus of b-catenin also binds several co-activators,including the histone acetylase CREB binding protein (CBP), Hyrax, and Brahma-relatedgene (Brg-1). Reprinted with permission from (4) . Copyright Elsevier (2006) .

Following its entry into the nucleus, β-catenin binds to theN-terminus of Tcf and converts Tcf into a potent transcriptional activator(32 – 34) . The resulting transient activation of the Wnt target geneprogram signals the final step in the Wnt signalling pathway. β-cateninachieves this by displacing the Groucho/TLE co-repressor proteinsfrom Tcf (35) and efficiently recruiting a variety of proteins capable ofeffecting changes in local chromatin structure to the Wnt target genes(31) (Fig. 1.3 ). Many of these co-activator proteins, such as the histoneacetylase CREB-binding protein (CBP), Brahma-related gene (BRG)-1(a component of the SWI/SNF chromatin remodelling complex),and Hyrax interact directly with the C-terminus of β-catenin. Another protein, Pygopus, indirectly binds the N-terminus of β-cateninvia a common binding partner, Bcl9. The precise role of the β-catenin/Bcl9/Pygopus complex is somewhat controversial; one lineof evidence suggests that it facilitates the nuclear import/retentionof β-catenin (36) , whilst another study supports a direct role for thiscomplex in enhancing the ability of β-catenin to activate Wnt targetgenes (37) .

6. Biological Consequences of Wnt Signalling

Activation of the Wnt pathway at the cell surface is ultimatelytranslated into a biological response through activation of a selectset of Tcf/β-catenin-responsive target genes. Of the currently estimated400 target genes present in the mammalian genome, onlya fraction of these are thought to be Wnt responsive at any giventime in a particular cell type (4) . This allows the transcriptionaloutput of the Wnt signal to be tailored to meet the specific needsof a given cell type. For example, in the epithelium of intestinalcrypts, Wnt signalling drives both the proliferation of stem/progenitorcells (38 , 39) and terminal differentiation of Paneth cells(40) . The advent of microarray technologies has facilitated theidentification of many of these Wnt target genes (41 – 43) . Thishas provided important clues as to how Wnt signalling influencessuch diverse biological events as cell proliferation, cell fate specification,terminal differentiation, and cell migration. For example,c-Myc and cyclin D1 are considered to be potent activators of cellproliferation, whilst other target genes encoding the guidancereceptors EphB2 and EphB3 are instrumental in cell positioningwithin tissues such as the intestine (44) .

Given the wide-ranging influence of Wnt signalling on such adiverse set of biological processes, it is perhaps not too surprisingthat loss of proper regulation of this signalling activity can havedisastrous consequences for embryonic development and tissuerenewal in adults (4 , 39) . For proof of this, we only have to lookat the large variety of severe phenotypes in multiple tissues andorgans caused by artificially induced loss of Wnt signalling componentsin flies, frogs, fish, and mice (7) . In adults, Wnt signallingremains essential throughout life for driving tissue renewalin organs such as the intestine and skin (39) . In these rapidlyself-renewing tissues, Wnt signalling is instrumental in maintainingproliferation of stem cell populations and driving expansionof new epithelial cell precursors. More recent evidence suggeststhat Wnt signalling is likely to have a more general role in maintainingstem cell populations in a variety of tissues, including thehematopoietic system (39) . This raises the attractive possibility ofstem cells expressing unique Wnt target genes that could be usedas specific markers for identifying and ultimately isolating thesecells from a variety of tissues.

However, it is also becoming increasingly apparent that Wntpathway mutations can occur. These mutations upset the homeostaticbalance in self-renewing tissues and cause a variety of diseasesincluding bone defects and cancer. In the early stages ofcolon cancer for example, mutations frequently occur in eitherAPC or β-catenin that cause constitutive activation of the Wntpathway and promote uncontrolled cell proliferation (29 , 45) .Deregulation of the Wnt pathway is also associated with severalother types of human cancer and disease (4 , 46) . This has fuelledefforts to try and develop specific inhibitors of the Wnt pathwayfor use as cancer therapeutics, although serious challenges remainto be overcome before this can become a reality (5) .

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