Modulating Wnt signaling at the root: Porcupine and Wnt acylation

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Abstract

Communication between cells occurs through secreted molecules, among which Wnt ligands play a critical role in balancing cell proliferation, differentiation and cellular homeostasis. The action of Wnt signaling can be modulated at several levels, including posttranslational modification of the Wnt ligands, whose acylation is critical for biological activity. At least three enzymes are necessary for Wnt acylation/deacylation: stearoyl CoA desaturase (SCD), porcupine (PORCN) and Notum. At the endoplasmic reticulum (ER), SCD provides the monounsaturated fatty acid to PORCN, which adds it to the Wnt ligand; at the extracellular matrix, the fatty acid is removed by Notum. Obviously, the interplay between these enzymes will define Wnt signaling ligand secretion and activity. Excessive activation of Wnt signaling has been observed in a variety of solid tumors, which has led the pharmaceutical industry to develop specific inhibitors for this pathway that mainly target PORCN, some of which are in early clinical trials. In the central nervous system (CNS), Wnt signaling activation has been shown to have a neuroprotective effect, and conversely, its inhibition induces neurodegeneration, which implies that the inhibition of PORCN in cancer therapies should be used with caution, and the cognitive performance of the patient should be monitored during treatment. This review collects information about the PORCN enzyme in relation to its role in the Wnt pathway through the acylation of Wnt ligands, its inhibition by drugs in the treatment of some cancers, and its putative modulation in the treatment of neurodegenerative diseases.

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”.

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