Activation of Wnt Signaling by Chemically Induced Dimerization of LRP5 Disrupts Cellular Homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion

A composite MyD88/CD40 switch synergistically activates mouse and human dendritic cells for enhanced antitumor efficacy

The in vivo therapeutic efficacy of DC-based cancer vaccines is limited by suboptimal DC maturation protocols. Although delivery of TLR adjuvants systemically boosts DC-based cancer vaccine efficacy, it could also increase toxicity. Here, we have engineered a drug-inducible, composite activation receptor for DCs (referred to herein as DC-CAR) comprising the TLR adaptor MyD88, the CD40 cytoplasmic region, and 2 ligand-binding FKBP12 domains. Administration of a lipid-permeant dimerizing ligand (AP1903) induced oligomerization and activation of this fusion protein, which we termed iMyD88/CD40. AP1903 administration to vaccinated mice enabled prolonged and targeted activation of iMyD88/CD40-modified DCs. Compared with conventionally matured DCs, AP1903-activated iMyD88/CD40-DCs had increased activation of proinflammatory MAPKs. AP1903-activated iMyD88/CD40-transduced human or mouse DCs also produced higher levels of Th1 cytokines, showed improved migration in vivo, and enhanced both antigen-specific CD8+ T cell responses and innate NK cell responses. Furthermore, treatment with AP1903 in vaccinated mice led to robust antitumor immunity against preestablished E.G7-OVA lymphomas and aggressive B16.F10 tumors. Thus, the iMyD88/CD40 unified “switch” effectively and safely replaced exogenous adjuvant cocktails, allowing remote and sustained DC activation in vivo. DC “licensing” through iMyD88/CD40 may represent a mechanism by which to exploit the natural synergy between the TLR and CD40 signaling pathways in DCs using a single small molecule drug and could augment the efficacy of antitumor DC-based vaccines

Dimerized-iLRP5 localizes to disheveled-containing punctate structures.

<p>(<b>A, B</b>) Subcellular localization of dimerized LRP5c and disheveled. 293 cells were transfected with 1 µg each of plasmids expressing drug-binding domains fused with GFP (F3-GFP) or GFP-LRP5c (F3-GFP-LRP5c) with (B) or without (A) RFP-fused disheveled construct, Dvl-RFP. Twenty-four hours after transfection, 100 nM FK1012 was added for an additional 24 hours to induce F3-LRP5c dimerization. After fixation, cells were labeled with cholera toxin B (CTx-B-TRITC) (A) and examined by fluorescence microscopy. (<b>C, D</b>) Independent role of iLRP5 and disheveled in β-catenin activation. 293 cells were transfected with 1 µg TCF-SEAP reporter along with 0.5 µg of iLRP5 and the indicated amounts (µg) of Axin or dominant negative disheveled (Dix) constructs (C) or 0.5 µg disheveled construct (D). Twenty-four hours after transfection, 100 nM CID was added and SEAP activity was measured after 24 hours. Data is representative of at least three independent experiments (A, B). Error bars indicate mean ± S.D. of triplicate measurements (C. D).</p

Mammary gland transplant assay using Ubi-Cat-derived mammary cells.

<p>Ubi-Cat mammary cells were isolated from 6–8 week adult female Ubi-Cat mice. 5×10³ WT or Ubi-Cat mammary cells were transplanted into virgin adult recipients. WT and untreated Ubi-Cat cells demonstrated normal mammary outgrowth 8 weeks after transplant. Activation of iLRP via AP20187 resulted in formation of hyperplastic mammary ducts in 4 of 6 grafts. Two of six CID-treated grafts formed mammary tumors.</p

Axin is a critical downstream regulator of iLRP5 and interacts with iLRP5.

<p>(<b>A</b>) Effects of dimerization and membrane translocation of LRP5c on Wnt/β-catenin signaling. Activation of the TCF reporter in 293 cells transiently transfected with 1 µg each TCF-SEAP reporter and M_F-LRP5c (○), M_F-FRB_l2+F3-GFP (•), F3-LRP5c (□) or M_F-FRB_l2+F3-LRP5c (▪) expression plasmids is shown. After 24 hours, cell aliquots were treated with CID and suboptimal (5 nM) Rap-B, and SEAP activity was measured after 24 hours. (<b>B</b>) Effect of Axin on iLRP5-mediated β-catenin activation. 293 cells were transiently transfected with 1 µg each of TCF-SEAP and iLRP5 plasmids with our without myc-tagged Axin. After 24 hours, cells were treated with 100 nM CID (AP20187) or diluent alone, and SEAP activity was measured after an additional 24 hours. Protein expression levels were determined by anti-myc and anti-HA blotting (inset). (<b>C</b>) Dimerization-dependent interaction of Axin and iLRP5. 293 cells were transfected with 1 µg of indicated combinations of iLRP5 and Axin. After treatment with 100 nM homodimer (AP20187) for 24 hours (lane 2, 4), whole cell lysates were immunoprecipitated with anti-myc antibody (myc). Co-immunoprecipitation of LRP5c was analyzed by anti-HA blot (HA). Backbone (F_v′-F_vls-HA) or iLRP5 (F_v′-F_vls-iLRP5-HA) protein expression was detected by anti-HA blot (Input: HA). All data are representative of at least two (panel A) or three (panels B–C) independent experiments with similar results. Error bars (B) represent S.D. of triplicate measurements.</p

<i>In vitro</i> analysis of iLRP5 activation in prostate B/SCs.

<p>(<b>A</b>) qRT-PCR analysis of Wnt target genes. LSCs were cultured in Matrigel for 6 days with or without CID (100 nM). Cells were isolated and RNA was collected for cDNA synthesis and qRT-PCR analysis. CID-mediated iLRP5 activation promotes Wnt target genes expression. Error bars represent StDev of 3 experiments, normalized to 18S RNA (<b>B</b>) Prolonged (16 days) activation of iLRP5 promotes enlargement of prostaspheres. Control spheres grew to maximum size of 150–200 µm; however, CID-treated spheres grew to 300–400 µm. Bar = 100 µm. (<b>C</b>) Analysis of p63 and β-catenin expression in prostasphere cross-sections. iLRP5 activation enhances β-catenin stability. CID-treated spheres reveal dispersed distribution of p63⁺ cells within all layers of prostasphere. Bar = 100 µm.</p

A novel region of LRP5 (₁₅₁₆MFYSSNIPATVRPYRPY₁₅₃₁) is required for Wnt signaling.

<p>(<b>A</b>) Cross-species analysis of the novel LRP5 (₁₅₁₆MFYSSNIPATVRPYRPY₁₅₃₁) sequence. (<b>B</b>) Membrane targeting of the cytoplasmic moiety of LRP5 (M_F-LRP5) and three variants containing mutations of possible phosphorylation sites: (1) YSS→FAA, (2) YRPY→FLPF and (3) YSS→FAA and YRPY→FLPF. The mutants have reduced Wnt pathway inducibility. Western blot indicates comparable protein expression from respective TCF-SEAP assay. (<b>C</b>) Comparison of iLRP5 and iLRP5 mutants. YSS→FAA and YRPY→FLPF mutants have lower TCF-SEAP activity. The double mutant fails to activate Wnt signaling. Western blot indicates protein expression from respective TCF-SEAP assay. (B and C), Data representative of two separate experiments performed in triplicates.</p

Two distinct regulatory sites in LRP5c are critical for function and localization of iLRP5.

<p>(<b>A</b>) (left) Schematic of LRP5c deletion mutants (iLRP5 Δ1–5) comprised of synthetic ligand-binding domains (F_v′-F_vls). Numbers indicate N-terminal amino acid position of each deletion mutant. Red and black lines represent indicated sequences and gray lines represent PPPSP motifs, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030814#pone.0030814-Davidson1" target="_blank">[24]</a>. (right) Domain analysis of LRP5c on Wnt/β-catenin signaling. 293 cells were transiently transfected with 1 µg each of indicated constructs along with TCF-SEAP reporter. After 24 hours, cell aliquots were treated with indicated concentrations of AP20187, and SEAP activity was measured after an additional 24-hour incubation. Data represent two independent experiments with similar results. (<b>B</b>) Role of novel and CK1 sites in the interaction of Axin and iLRP5. 293 cells were transfected with 1 µg each of indicated mutants of iLRP5 along with an Axin expression construct. After 24-hour treatment with 100 nM AP20187 (+CID), Axin was immunoprecipitated from whole cell lysates with anti-myc antibody (α-myc). Co-immunoprecipitation of LRP5c was analyzed by anti-HA blot (α-HA). Amount of iLRP5 mutant expression was determined by HA blot (Input: α-HA). (<b>C</b>) Subcellular localization of iLRP5-truncation mutants. 293 cells were transfected with 1 µg of indicated plasmids. Twenty-four hours after transfection, 100 nM FK1012 was added for an additional 24 hours to induce dimerization. After fixation, cells were examined by fluorescence microscopy.</p