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-catenin17–19.

Wnt /β-Catenin signaling pathway

Image 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 diseases32,33.

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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 References

  1. Valenta, T., Hausmann, G., and Basler, K. (2012) The many faces and functions of β-catenin. EMBO J. 31, 2714–2736.
  2. Clevers, H., and Nusse, R. (2012) Wnt/β-catenin signaling and disease. Cell 149, 1192–1205.
  3. Takada, R., Satomi, Y., Kurata, T., Ueno, N., Norioka, S., Kondoh, H., Takao, T., and Takada, S. (2006) Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev. Cell 11, 791–801.
  4. Grzeschik, K.-H., Bornholdt, D., Oeffner, F., König, A., del Carmen Boente, M., Enders, H., Fritz, B., Hertl, M., Grasshoff, U., Höfling, K., Oji, V., Paradisi, M., Schuchardt, C., Szalai, Z., Tadini, G., Traupe, H., and Happle, R. (2007) Deficiency of PORCN, a regulator of Wnt signaling, is associated with focal dermal hypoplasia. Nat. Genet. 39, 833–835.
  5. Port, F., Kuster, M., Herr, P., Furger, E., Bänziger, C., Hausmann, G., and Basler, K. (2008) Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nat. Cell Biol. 10, 178–185.
  6. van den Heuvel, M., Nusse, R., Johnston, P., and Lawrence, P. A. (1989) Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication. Cell 59, 739–749.
  7. Gong, Y., Bourhis, E., Chiu, C., Stawicki, S., DeAlmeida, V. I., Liu, B. Y., Phamluong, K., Cao, T. C., Carano, R. A. D., Ernst, J. A., Solloway, M., Rubinfeld, B., Hannoush, R. N., Wu, Y., Polakis, P., and Costa, M. (2010) Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies. PloS One 5, e12682.
  8. Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L., and Wu, D. (2001) Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7, 801–809.
  9. Zeng, X., Tamai, K., Doble, B., Li, S., Huang, H., Habas, R., Okamura, H., Woodgett, J., and He, X. (2005) A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438, 873–877.
  10. Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., Glinka, A., and Niehrs, C. (2005) Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438, 867–872.
  11. Bovolenta, P., Esteve, P., Ruiz, J. M., Cisneros, E., and Lopez-Rios, J. (2008) Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J. Cell Sci. 121, 737–746.
  12. Ellwanger, K., Saito, H., Clément-Lacroix, P., Maltry, N., Niedermeyer, J., Lee, W. K., Baron, R., Rawadi, G., Westphal, H., and Niehrs, C. (2008) Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density. Mol. Cell. Biol. 28, 4875–4882.
  13. Shimomura, Y., Agalliu, D., Vonica, A., Luria, V., Wajid, M., Baumer, A., Belli, S., Petukhova, L., Schinzel, A., Brivanlou, A. H., Barres, B. A., and Christiano, A. M. (2010) APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature 464, 1043–1047.
  14. Xu, Q., Wang, Y., Dabdoub, A., Smallwood, P. M., Williams, J., Woods, C., Kelley, M. W., Jiang, L., Tasman, W., Zhang, K., and Nathans, J. (2004) Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895.
  15. de Lau, W., Barker, N., Low, T. Y., Koo, B.-K., Li, V. S. W., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P. J., van de Wetering, M., Stange, D. E., van Es, J. E., Guardavaccaro, D., Schasfoort, R. B. M., Mohri, Y., Nishimori, K., Mohammed, S., Heck, A. J. R., and Clevers, H. (2011) Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297.
  16. Aberle, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997) beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 16, 3797–3804.
  17. Kramps, T., Peter, O., Brunner, E., Nellen, D., Froesch, B., Chatterjee, S., Murone, M., Züllig, S., and Basler, K. (2002) Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 109, 47–60.
  18. Parker, D. S., Jemison, J., and Cadigan, K. M. (2002) Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila. Dev. Camb. Engl. 129, 2565–2576.
  19. Thompson, B., Townsley, F., Rosin-Arbesfeld, R., Musisi, H., and Bienz, M. (2002) A new nuclear component of the Wnt signalling pathway. Nat. Cell Biol. 4, 367–373.
  20. He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Identification of c-MYC as a target of the APC pathway. Science 281, 1509–1512.
  21. Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T., and Clevers, H. (1999) Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1. Science 285, 1923–1926.
  22. Filali, M., Cheng, N., Abbott, D., Leontiev, V., and Engelhardt, J. F. (2002) Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter. J. Biol. Chem. 277, 33398–33410.
  23. He, T. C., Chan, T. A., Vogelstein, B., and Kinzler, K. W. (1999) PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99, 335–345.
  24. Mann, B., Gelos, M., Siedow, A., Hanski, M. L., Gratchev, A., Ilyas, M., Bodmer, W. F., Moyer, M. P., Riecken, E. O., Buhr, H. J., and Hanski, C. (1999) Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc. Natl. Acad. Sci. U. S. A. 96, 1603–1608.
  25. Crawford, H. C., Fingleton, B. M., Rudolph-Owen, L. A., Goss, K. J., Rubinfeld, B., Polakis, P., and Matrisian, L. M. (1999) The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors. Oncogene 18, 2883–2891.
  26. Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H., Schlag, P. M., Birchmeier, W., and Behrens, J. (2002) Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184–1193.
  27. Conacci-Sorrell, M. E., Ben-Yedidia, T., Shtutman, M., Feinstein, E., Einat, P., and Ben-Ze’ev, A. (2002) Nr-CAM is a target gene of the beta-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis. Genes Dev. 16, 2058–2072.
  28. Miwa, N., Furuse, M., Tsukita, S., Niikawa, N., Nakamura, Y., and Furukawa, Y. (2001) Involvement of claudin-1 in the beta-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers. Oncol. Res. 12, 469–476.
  29. Zhang, X., Gaspard, J. P., and Chung, D. C. (2001) Regulation of vascular endothelial growth factor by the Wnt and K-ras pathways in colonic neoplasia. Cancer Res. 61, 6050–6054.
  30. Kahler, R. A., and Westendorf, J. J. (2003) Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2-dependent transcriptional activation of the osteocalcin promoter. J. Biol. Chem. 278, 11937–11944.
  31. Jamora, C., DasGupta, R., Kocieniewski, P., and Fuchs, E. (2003) Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422, 317–322.
  32. Andl, T., Reddy, S. T., Gaddapara, T., and Millar, S. E. (2002) WNT signals are required for the initiation of hair follicle development. Dev. Cell 2, 643–653.
  33. Gat, U., DasGupta, R., Degenstein, L., and Fuchs, E. (1998) De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95, 605–614.
  34. De Boer, J., Wang, H. J., and Van Blitterswijk, C. (2004) Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells. Tissue Eng. 10, 393–401.
  35. Nostro, M. C., Cheng, X., Keller, G. M., and Gadue, P. (2008) Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell 2, 60–71.
  36. Zhao, C., Blum, J., Chen, A., Kwon, H. Y., Jung, S. H., Cook, J. M., Lagoo, A., and Reya, T. (2007) Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell 12, 528–541.
  37. Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G., and Birchmeier, W. (2001) beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105, 533–545.
  38. Salinas, P. C. (2007) Modulation of the microtubule cytoskeleton: a role for a divergent canonical Wnt pathway. Trends Cell Biol. 17, 333–342.
  39. Yang, K., Wang, X., Zhang, H., Wang, Z., Nan, G., Li, Y., Zhang, F., Mohammed, M. K., Haydon, R. C., Luu, H. H., Bi, Y., and He, T.-C. (2016) The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies. Lab. Investig. J. Tech. Methods Pathol. 96, 116–136.
  40. Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Clevers, H., Vogelstein, B., and Kinzler, K. W. (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275, 1787–1790.
  41. Bass, A. J., Lawrence, M. S., Brace, L. E., Ramos, A. H., Drier, Y., Cibulskis, K., Sougnez, C., Voet, D., Saksena, G., Sivachenko, A., Jing, R., Parkin, M., Pugh, T., Verhaak, R. G., Stransky, N., Boutin, A. T., Barretina, J., Solit, D. B., Vakiani, E., Shao, W., Mishina, Y., Warmuth, M., Jimenez, J., Chiang, D. Y., Signoretti, S., Kaelin, W. G., Spardy, N., Hahn, W. C., Hoshida, Y., Ogino, S., Depinho, R. A., Chin, L., Garraway, L. A., Fuchs, C. S., Baselga, J., Tabernero, J., Gabriel, S., Lander, E. S., Getz, G., and Meyerson, M. (2011) Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion. Nat. Genet. 43, 964–968.
  42. Björklund, P., Akerström, G., and Westin, G. (2007) An LRP5 receptor with internal deletion in hyperparathyroid tumors with implications for deregulated WNT/beta-catenin signaling. PLoS Med. 4, e328.
  43. Takeda, H., Lyle, S., Lazar, A. J. F., Zouboulis, C. C., Smyth, I., and Watt, F. M. (2006) Human sebaceous tumors harbor inactivating mutations in LEF1. Nat. Med. 12, 395–397.
  44. Satoh, S., Daigo, Y., Furukawa, Y., Kato, T., Miwa, N., Nishiwaki, T., Kawasoe, T., Ishiguro, H., Fujita, M., Tokino, T., Sasaki, Y., Imaoka, S., Murata, M., Shimano, T., Yamaoka, Y., and Nakamura, Y. (2000) AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Nat. Genet. 24, 245–250.
  45. Liu, W., Dong, X., Mai, M., Seelan, R. S., Taniguchi, K., Krishnadath, K. K., Halling, K. C., Cunningham, J. M., Boardman, L. A., Qian, C., Christensen, E., Schmidt, S. S., Roche, P. C., Smith, D. I., and Thibodeau, S. N. (2000) Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling. Nat. Genet. 26, 146–147.
  46. Kinzler, K. W., Nilbert, M. C., Su, L. K., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., and McKechnie, D. (1991) Identification of FAP locus genes from chromosome 5q21. Science 253, 661–665.