A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities

Abstract

Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.

ERAD‐dependent control of the Wnt secretory factor Evi

“…Cells were harvested 48 h after transfection. The following expression constructs were used: pSpCas9(BB)‐2A‐Puro (px459; Addgene #48139; Ran et al , 2013), psPAX2 (Addgene #12260; D. Trono), pMD2.G‐VSVG (Addgene #12259; D. Trono), TCF4/Wnt‐Firefly Luciferase (Demir et al , 2013), Actin‐Renilla Luciferase (Nickles et al , 2012), pcDNA3‐Wnt11 (Addgene #35922; Najdi et al , 2012), pcDNA3‐Wnt10B, (Addgene #35921; Najdi et al , 2012), pcDNA3‐Wnt16 (Addgene #35923; Najdi et al , 2012), pcDNA3‐Wnt7B (Addgene #35915; Najdi et al , 2012), pcDNA3‐Wnt8 (Addgene #35916; Najdi et al , 2012), pcDNA3‐Wnt5A (Addgene #35911; Najdi et al , 2012), pcDNA3‐Wnt3A (Addgene #35908; Najdi et al , 2012), pcDNA3‐Wnt3 (Addgene #35909; Najdi et al , 2012), pcDNA3.2‐Wnt3A‐V5 (Addgene #43810; MacDonald et al , 2014), pcDNA3‐IGFBP5‐V5 (Addgene #11608; S. Johnson), pcDNA3‐EGF‐Myc (Addgene #11601; Nguyen et al , 2000), VCP(wt)‐EGFP (Addgene #23971; Tresse et al , 2010), VCP(DKO)‐EGFP (Addgene #23974; Tresse et al , 2010), pCMV6‐Myc‐DDK‐tagged PORCN (Origene # RC223764), sLuc (Katanaev et al , 2008), pCS2(+)‐mWnt3A (C. Niehrs), pCS2(+)‐Dvl3 (C. Niehrs), pCS2(+)‐mWnt3A‐S209A (Kumar et al , 2014), and β‐Catenin‐YFP (Stemmer et al , 2008). To generate C‐terminally FLAG‐HA‐tagged CGRRF1 and Hrd1, the FLAG‐HA sequence was inserted into the pcDNA5‐FRT/TO vector (Invitrogen).…”

Canonical and noncanonical Wnt signaling in neural stem/progenitor cells

“…Adding to this complexity, the Wnt response can differ depending on cell context and on the repertoire of Wnt receptors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 expressed [25,26], further underlining the importance of determining the "Wnt status" of cells. ROR2, for example, has been shown to activate phosphatidylinositol-3 kinase (PI3K), which in turn activates JNK and its associated transcription factors c-Jun and ATF2 in Xenopus [27,28].…”

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

“…13, 29-31). Thus, inhibition of PORCN blocks the secretion and activity of all human Wnts (32,33). Small-molecule PORCN inhibitors circumvent some of the limitations of the recombinant protein/monoclonal antibody approaches, and will inhibit both canonical and noncanonical signaling pathways.…”