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A Uniform Human Wnt Expression Library Reveals a Shared Secretory Pathway and Unique Signaling Activities

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Differentiation 84 (2012) 203–213 Contents lists available at SciVerse ScienceDirect Differentiation journal homepage: www.elsevier.com/locate/diff A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities Rani Najdi a, Kyle Proffitt b, Stephanie Sprowl a, Simran Kaur b, Jia Yu b, Tracy M. Covey b, David M. Virshup b,1, Marian L. Waterman a,n a Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA b Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore article info abstract Article history: Wnt ligands are secreted morphogens that control multiple developmental processes during embry- Received 24 April 2012 ogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Accepted 16 June 2012 Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these Available online 9 July 2012 signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We Keywords: have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing Wnt signaling epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the b-catenin mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of Wnt modification and secretion the 19 Wnts activate b-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of b-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 b-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function. & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. 1. Introduction fates including cell surface binding, local diffusion, binding to lipoprotein carriers, and secretion in vesicles (Banziger et al., 2006; The Wnt signaling pathways regulate key networks during Bartscherer et al., 2006). Once secreted, these Wnts are further both embryonic development and adult tissue homeostasis. These regulated by interaction with a variety of extracellular regulators signaling outcomes play a major role in the regulation of cell fate including members of the decoy receptor family SFRP. In total, there decisions such as proliferation, survival and differentiation. are 19 human Wnts that regulate numerous biological processes via Because of its well-documented contributions to these processes, diverse receptors and signaling pathways. One well-studied prop- misregulation of Wnt signaling contributes to a variety of human erty of many Wnts is their ability to stabilize b-catenin, an activity diseases, ranging from defects of development and cancers of the initiated by binding to LRP5/6 and Frizzled family cell surface colon, breast and skin, to diseases associated with defects of the receptors (van Amerongen and Nusse, 2009). Importantly, Wnts eye, bone and heart (Klaus and Birchmeier, 2008). also signal through Frizzled and additional classes of cell surface At the center of this complex network are Wnt ligands, a highly receptors including receptor tyrosine kinases, to regulate diverse conserved family of cysteine-rich secreted morphogens. Wnt pro- b-catenin-independent pathways, including the Planar Cell Polarity teins are synthesized in the endoplasmic reticulum (ER) where the (PCP) and the Wnt/calcium pathways (Najdi et al., 2011). membrane bound O-acyltransferase porcupine (PORCN) catalyzes Given the complexity of Wnt biology in humans, many ques- their palmitoylation (Takada et al., 2006; Willertetal.,2003). tions regarding the roles and activities of various Wnts remain. Modified Wnt ligands are carried to the plasma membrane by The classification of Wnts has been largely based on their binding in a palmitoylation dependent manner to the carrier protein signaling properties across different systems rather than direct Wntless (WLS). At the plasma membrane, Wnts undergo a variety of comparison among Wnts of the same species in a single cell type. For example, WNT1 and 3A are traditionally considered to be potent activators of Wnt/b-catenin signaling while other Wnts n Corresponding author. Tel.: þ19498243096; fax: þ19498248598. such as 5A and 11 are known to activate b-catenin-independent E-mail addresses: [email protected] (D.M. Virshup), pathways. However, recent reports have demonstrated that [email protected] (M.L. Waterman). 1 Tel.: þ65 6516 6954; fax: þ65 6221 2402. WNT5A can activate Wnt/b-catenin signaling depending on the 0301-4681/$ - see front matter & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) http://dx.doi.org/10.1016/j.diff.2012.06.004 204 R. Najdi et al. / Differentiation 84 (2012) 203–213 receptors expressed at the plasma membrane. Furthermore, both primer (50-CGCGCCGACCCAGCTTTCTTG-30) and destVect121R1 anti- WNT5A and 11 have been shown to heterodimerize to activate sense primer (50-CGGTACGCGTAGAATCGAGACCG-30). Melting and b-catenin-dependent signaling (Cha et al., 2009; Mikels and annealing temperatures were 95 1Cand601C. Nusse, 2006). Similarly, WNT3A has been shown to induce morphogenetic changes by activating Rho-associated kinase or c-Jun N-terminal kinase, depending on cell type (Endo et al., 2.3. Western analysis 2008; Kishida et al., 2004). These data confirm that signaling is complex and will be dependent on multiple factors beyond single For detection of WNT expression and secretion, HEK293 or ligand/receptor interactions. However, the tools to compare the NIH3T3 cells were cultured in DMEM media with 10% FBS activity of human Wnts have been lacking. (Cellgro) and plated at a density of 1000,000 cells per plate in One apparently common pathway for Wnt signaling is that of 10 cm dishes. Cells were transfected using Bio T transfection Wnt production, processing and secretion. However, while a reagent (Bioland Scientific) 24 h post plating with plasmids con- small number of Wnts have been examined for posttranslational taining V5-tagged Wnts (pcDNA3.2/V5-DEST; 5 mg) or untagged modification and secretion, other mammalian Wnt ligands have WNT 1 and 3A (pcDNA3.2/V5-DEST; 5 mg). Conditioned media and not been characterized. This information gap becomes more cell lysates were harvested 48 h post transfection. The conditioned important to address as inhibitors of Wnt secretion enter clinical media was concentrated using StrataClean resin (Agilent Technol- trials (www.clinicaltrials.gov). These questions and the lack of ogies). Media, lysates and a V5 protein standard (Recombinant understanding of Wnt signaling properties have emphasized the Yeast Calmodulin Kinase Array Control Protein, Invitrogen) were requirement for a full standardized set of Wnt expression plas- separated by 10% SDS-PAGE and transferred to a nitrocellulose mids. Such a set would allow for a direct side-by-side comparison membrane (Whatman). For non-phospho b-catenin and phospho- of Wnt processing, secretion and signaling. For this reason, we LRP6 detection, HEK293 cells were plated at a density of 200,000 started the ‘‘Open Source Wnt’’ plasmid depository and cloned cells per well in 6-well plates and transfected using Bio T 24 h open reading frame sequences for all 19 human Wnts into the later with plasmids containing untagged Wnts (pcDNA3.2/V5- same expression plasmid backbone. Using this plasmid set, we DEST; 1 mg). Cell lysates were harvested 24 h post transfection, have been able to analyze and compare the expression levels and separated by 10% SDS-PAGE and transferred to a nitrocellulose the efficiency of secretion of all human Wnts in a defined set of membrane (Amersham Biosciences). Blots were blocked in 5% cell lines, revealing valuable insight on their processing, their milk for 30 min and hybridized with primary antibodies against signaling potential and their accumulation in conditioned media. V5 (1:5000 dilution, Invitrogen), Actin (I-19, 1:500, Santa Cruz), We show that, dependent on cell type, up to 14 out of the 19 Lamin A/C (1:5000, Cell Signaling), WNT1 (N2C3, 1:1300, Gene- Wnts are capable of signaling through the b-catenin-dependent Tex), WNT3A (1:250, R&D Systems), non-phospho b-catenin pathway to activate the Super8XTOPflash reporter. Wnt/b-catenin (Ser33/37/Thr41, 1:500, Cell Signaling), phospho-LRP6 (Ser1490, signaling is broadly reduced by the secreted Wnt inhibitors Dkk-1 1:1000, Cell Signaling), or b-Tubulin (TUBB1, 1:1000, GeneTex) and the SFRP1 and 3 (Secreted Frizzled-Related Proteins). Activa- overnight at 4 1C. After hybridization, the blots were washed and tion of the Super8XTOPflash reporter, with a few exceptions, is hybridized with anti-rabbit IgG-HRP (1:5000, Amersham Bios- more sensitive than, but tracks with, LRP6 phosphorylation and ciences), anti-mouse IgG-HRP (1:5000, Amersham Biosciences), the stabilization of b-catenin. Finally, we find that PORCN and anti-rat IgG-HRP (1:50,000, Jackson ImmunoResearch) or Bovine WLS are essential elements of a common Wnt secretion pathway, anti-goat IgG-HRP (1:15,000, Santa Cruz) for 2 h at RT. The ECL as all assessable Wnts require PORCN-dependent palmitoylation reaction was performed according to the manufacturer’s protocol to bind to the carrier protein WLS and to signal through b- (Thermo Scientific) and blots were visualized using the LAS-4000 catenin-dependent and independent pathways. Fujifilm imaging system. 2. Materials and methods 2.4. Luciferase assays 2.1. Wnt cloning HEK293 or NIH3T3 cells were cultured in DMEM media with 10% FBS and plated at a density of 200,000 cells per well in 6-well plates. Human WNT cDNAs were PCR-amplified twice: one reaction Cells were transfected 24 h post plating, using BioT, with Super8- using Wnt-specific sense primers and ‘‘STOP’’ antisense primers, XTOPflash reporter plasmid (0.1 mg; kind gift from Dr.
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