Modulating Wnt signaling at the root: Porcupine and Wnt acylation
Introduction
Wnt proteins were discovered in invertebrates and vertebrates in the 1980s by two groups. In 1980, Christiane Nüsslein-Volhard and Eric Wieschaus, in search of gene mutations affecting the segmental pattern of the Drosophila larva, discovered Wnt/Wingless (wg) (Nüsslein-Volhard & Wieschaus, 1980). Two years later, Nusse and Varmus, while exploring genomic sites where DNA integration might induce breast cancer in mice, discovered Int1 (Nusse & Varmus, 1982), which was later shown to be the same Wg gene previously described in Drosophila (Nüsslein-Volhard & Wieschaus, 1980). Therefore, the name Wnt is derived from a combination of Wg and Int (Nusse, 1991).
Wnt ligands are involved in a number of cellular processes during development, including cell fate, proliferation, polarity, cell migration, and the homeostasis of mature tissues (Nusse, 2012; Nusse & Varmus, 2012; Willert et al., 2003). In humans, 19 Wnt proteins have been described, and Wnt ligands are recognized by seven transmembrane-spanning receptors, including Frizzled (Fzd) and its coreceptor Low-density lipoprotein receptor-related protein 5/6 (LRP5/6), which have ten and two isoforms in humans, respectively. The ligand, its receptor and the scaffold protein Disheveled (Dvl) form a signalosome that transduces Wnt signaling intracellularly (Acebron & Niehrs, 2016; Bilic et al., 2007).
Wnt ligands are secreted short-and long-range action molecules that can trigger two intracellular pathways: the canonical and the noncanonical (Gordon & Nusse, 2006). In the canonical pathway, β-catenin is stabilized in the cytoplasm through inhibition of the β-catenin degradation complex. Then, β-catenin is free to enter the nucleus, where it activates Wnt-regulated genes through its interaction with T-cell factor (TCF) family of transcription factors. This pathway branches to trigger a variety of other processes that are independent of β-catenin (noncanonical Wnt signaling). Two noncanonical pathways that have been well characterized are (i) planar cell polarity (PCP) signaling, which leads to the activation of the small GTPases RAS homologue gene-family member A (RHOA) and RAC1, which in turn, activates the stress kinase Jun N-terminal kinase (JNK) and RHO-associated coiled-coil-containing protein kinase 1 (ROCK) to instigate remodeling of the cytoskeleton and changes in cell adhesion and motility, and (ii) the Wnt-calcium pathway, in which G proteins and phospholipases mediate a transient increase in cytoplasmic free calcium activating protein kinase C (PKC), calcium calmodulin mediated kinase II (CAMKII) and the phosphatase Calcineurin (Acebron & Niehrs, 2016; Habas, Kato, & He, 2001; Nusse, 2012; Rosso, Sussman, Wynshaw-Boris, & Salinas, 2005).
PORCN is a membrane-bound O-acyltransferase (MBOAT) that acylates Wnt molecules at specific sites, conferring functional activity on the Wnt protein family (Cho & Park, 2016; Clements, 2009; Hofmann, 2000). This acylation is critical for Wnt molecules to bind Wntless (Wls), a cargo receptor that allows transportation of the Wnt ligand from the Golgi apparatus to the cell surface (Bartscherer, Pelte, Ingelfinger, & Boutros, 2006; Galli, Zebarjadi, Li, Lingappa, & Burrus, 2016), and for efficient ligand binding to the Fzd receptor (Janda, Waghray, Levin, Thomas, & Garcia, 2012; Komekado, Yamamoto, Chiba, & Kikuchi, 2007)(Fig. 1). The presence of Wls in the Golgi apparatus of Drosophila Wg-producing cells is essential for Wnt secretion and activity (Port et al., 2008). Wls, traffics in a loop, i.e., it cycles between the Golgi and the plasma membrane; it returns to the Golgi by clathrin-mediated endocytosis in a mechanism that depends on the retromer (Fig. 1). In the Drosophila neuromuscular junction (NMJ), the release of Wnt1 and Wls occurs in association with exosome vesicles (Koles & Budnik, 2012). Interfering with this process impairs Wnt secretion (Bradley & Brown, 1990; Du et al., 2016; Herr & Basler, 2012; Papkoff & Schryver, 1990; Port et al., 2008).
Diverse drugs that recognize specific molecules involved in Wnt signaling have been used to decipher the operation of the Wnt pathway in normal and pathological conditions. For that purpose, drugs that act at three different levels of Wnt signaling have been designed: drugs that inhibit intracellular signaling at different molecular targets, drugs that interfere with the binding of the ligand to the receptor and drugs that inhibit the secretion of the Wnt ligand. The last group includes PORCN inhibitors (Blagodatski, Poteryaev, & Katanaev, 2014; Tapia-Rojas & Inestrosa, 2018a).
Here, we review the existing literature about Wnt acylation sites, PORCN isoforms, PORCN-specific inhibitors and their effects on neuronal health. We also present an open question about the potential of the modulation of Wnt signaling in the early treatment of neurodevelopmental and neurodegenerative diseases.
Section snippets
Wnt synthesis, posttranslational modification and secretion
Wnt molecules perform key roles during early development and throughout the adult life of an organism. They participate in cell proliferation, cell differentiation, cell migration, synaptic maturation and polarity. Functionally active Wnt proteins need to be modified posttranslationally to enable the Wnt ligand to be secreted and to bind to the Fzd receptor (Herr, Hausmann, & Basler, 2012). In eukaryote cells, after protein synthesis occurs, most of the proteins are delivered to the ER, where
Porcupine gene and protein
PORCN was first discovered in Drosophila during a screen of genes affecting patterning during development (van den Heuvel, Harryman-Samos, Klingensmith, Perrimon, & Nusse, 1993). PORCN belongs to a family of 16 evolutionarily conserved genes with predictable acyltransferase activity, the MBOAT family (Hofmann, 2000). It is located in the membrane of the ER and contains 11 predicted transmembrane domains (Fig. 3A) (Hofmann, 2000; Rios-Esteves, Haugen, & Resh, 2014) and a carboxy-terminal tail
Role of porcupine acylation in Wnt ligand properties and mechanism of action
The role of PORCN in Wnt signaling was first suggested during a genetic screening in Drosophila, where it was found that the mutation of its gene produces a phenotype similar to those generated by Wg mutations (Kadowaki, Wilder, Klingensmith, Zachary, & Perrimon, 1996; Manoukian, Yoffe, Wilder, & Perrimon, 1995; van den Heuvel et al., 1993). In PORCN mutant flies, Wg accumulated in ligand-producing cells, suggesting that PORCN was necessary for the processing and/or secretion of Wg (Kadowaki et
Specific drugs targeting PORCN
Dysregulation of the Wnt pathway has been associated with endocrine (Schinner, 2009), metabolic (Ackers & Malgor, 2018), inflammatory (Chilosi et al., 2003) and neurodegenerative diseases (Inestrosa & Arenas, 2010; Inestrosa, Montecinos-Oliva, & Fuenzalida, 2012; Inestrosa & Toledo, 2008). Moreover, Wnt signaling is known to be a critical pathway in oncogenic processes (Nusse & Varmus, 2012; Tsukamoto, Grosschedl, Guzman, Parslow, & Varmus, 1988; Zhan, Rindtorff, & Boutros, 2017), and
Wnt signaling targeting during neurodevelopment and neurodegeneration
Wnt signaling plays a role in several biological processes both in prokaryotes and in eukaryotes. There is a body of evidence that the canonical and noncanonical Wnt signaling pathways participate in different physiological aspects of synaptic differentiation, strength, and synaptic plasticity in later stages of development and in the mature organism (Dickins & Salinas, 2013; Dinamarca, Di Luca, Godoy, & Inestrosa, 2015; Inestrosa & Arenas, 2010; Oliva, Montecinos-Oliva, & Inestrosa, 2018).
Concluding remarks
It is widely accepted that Wnt signaling plays diverse roles in organ development and during animal adulthood, and its dysregulation is associated with certain cancers, neurodegenerative diseases, osteoporosis, and fibrosis (Fig. 6). It is also being considered for regenerative medicine because Wnt ligands provide a mechanism for signaling to modulate synaptic plasticity and brain function in later stages of development and in the mature organism, turning this pathway into an interesting
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgments
This study was supported by the Chilean grants: Basal Center of Excellence in Aging and Regeneration (AFB 170005) and FONDECYT NO #1160724 to N.C.I., and BMBF #20150065 to V.I.T. We also thank the Sociedad Química y Minera de Chile (SQM) for the special grant “The Role of Lithium in Human Health and Disease”.
References (139)
- et al.
β-Catenin-independent roles of Wnt/LRP6 signaling
Trends in Cell Biology
(2016) - et al.
Secretion of Wnt ligands requires Evi, a conserved transmembrane protein
Cell
(2006) - et al.
Glycogen synthase kinase-3beta immunoreactivity is reduced in the prefrontal cortex in schizophrenia
Neuroscience Letters
(2001) - et al.
Molecular cloning and initial characterization of the MG61/PORC gene, the human homologue of the Drosophila segment polarity gene porcupine
Gene
(2002) - et al.
Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons
The Journal of Biological Chemistry
(2008) - et al.
Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis
The American Journal of Pathology
(2003) - et al.
Palmitoylation in Alzheimer's disease and other neurodegenerative diseases
Pharmacological Research
(2016) - et al.
Structural correlates of cognition in dementia: Quantification and assessment of synapse change
Neurodegeneration
(1996) - et al.
The soluble extracellular fragment of neuroligin-1 targets Aβ oligomers to the postsynaptic region of excitatory synapses
Biochemical and Biophysical Research Communications
(2015) - et al.
Diverse chemical scaffolds support direct inhibition of the membrane-bound O-acyltransferase porcupine
The Journal of Biological Chemistry
(2012)
Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling
Cellular Signalling
Wnt-5a/JNK signaling promotes the clustering of PSD95 in hippocampal neurons
The Journal of Biological Chemistry
The role of glypicans in hedgehog signaling
Matrix Biology
Divergent effects of porcupine and Wntless on WNT1 trafficking, secretion, and signaling
Experimental Cell Research
Wnt signaling: Multiple pathways, multiple receptors, and multiple transcription factors
The Journal of Biological Chemistry
A potential vehicle for the spread of morphogens through epithelia
Cell
Wnt/frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1
Cell
Wnt lipid modifications: Not as saturated as we thought
Developmental Cell
Porcupine-mediated lipidation is required for Wnt recognition by Wls
Developmental Biology
Wnt secretion and signalling in human disease
Trends in Molecular Medicine
The use of porcupine inhibitors to target Wnt-driven cancers
Bioorganic & Medicinal Chemistry Letters
A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling
Trends in Biochemical Sciences
GSK-3 and the neurodevelopmental hypothesis of schizophrenia
European Neuropsychopharmacology
Amyloid-beta binds to the extracellular cysteine-rich domain of frizzled and inhibits Wnt/beta-catenin signaling
The Journal of Biological Chemistry
Increased expression of Wnt-1 in schizophrenic brains
Schizophrenia Research
Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome
Cell
Neurodevelopmental origins of bipolar disorder: iPSC models
Molecular and Cellular Neurosciences
Neocortical neurogenesis and the etiology of autism spectrum disorder
Neuroscience and Biobehavioral Reviews
Interrelationship of canonical and non-canonical Wnt signalling pathways in chronic metabolic diseases
Diabetes & Vascular Disease Research
Signaling across the synapse: A role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release
The Journal of Cell Biology
Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of wingless
Development (Cambridge, England)
Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome
Proceedings of the National Academy of Sciences US
The importance of Wnt signalling for neurodegeneration in Parkinson's disease
Biochemical Society Transactions
Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation
Science
Targeting the Wnt pathways for therapies
Molecular and Cellular Therapies
The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix
The EMBO Journal
Glypican-3 binds to frizzled and plays a direct role in the stimulation of canonical Wnt signaling
Journal of Cell Science
Frizzled-1 is involved in the neuroprotective effect of Wnt3a against Aβ oligomers
Journal of Cellular Physiology
Acyltransferases for secreted signalling proteins
Molecular Membrane Biology
Therapeutic targeting of tumor-derived R-Spondin attenuates β-catenin signaling and tumorigenesis in multiple cancer types
Cancer Research
Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer
Nature Chemical Biology
Discovery of pyridinyl acetamide derivatives as potent, selective, and orally bioavailable porcupine inhibitors
ACS Medicinal Chemistry Letters
Wnt-C59 arrests stemness and suppresses growth of nasopharyngeal carcinoma in mice by inhibiting the Wnt pathway in the tumor microenvironment
Oncotarget
Importance of PORCN and Wnt signaling pathways in embryogenesis
American Journal of Medical Genetics Part A
Abnormalities of Wnt signalling in schizophrenia--evidence for neurodevelopmental abnormality
Neuroreport
Wnt gradient formation requires retromer function in Wnt-producing cells
Science
Synapse loss in frontal cortex biopsies in Alzheimer's disease: Correlation with cognitive severity
Annals of Neurology
Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by beta-amyloid fibrils
Molecular Psychiatry
Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein
The EMBO Journal
Wnts in action: From synapse formation to synaptic maintenance
Frontiers in Cellular Neuroscience
Cited by (61)
Metabolic dependencies and targets in ovarian cancer
2023, Pharmacology and TherapeuticsNovel PORCN inhibitor WHN-88 targets Wnt/β-catenin pathway and prevents the growth of Wnt-driven cancers
2023, European Journal of PharmacologyCanonical WNT signaling pathway in cancer stem cells and potential inhibitors of therapeutic importance
2023, Cancer Stem Cells and Signaling PathwaysInhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis
2024, Clinical Cancer ResearchAdvances in regulation and function of stearoyl-CoA desaturase 1 in cancer, from bench to bed
2023, Science China Life Sciences