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• W2763150312 abstract "•ANGPTL4 is a Wnt signaling antagonist that binds syndecans•ANGPTL4 promotes turnover of the Wnt co-receptor LRP6•ANGPTL4 is a Spemann organizer gene in Xenopus and promotes notochord development Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/β-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease. Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/β-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease. The Wnt/β-catenin signaling pathway controls numerous developmental processes and is implicated in stem cell biology and human disease (Holland et al., 2013Holland J.D. Klaus A. Garratt A.N. Birchmeier W. Wnt signaling in stem and cancer stem cells.Curr. Opin. Cell Biol. 2013; 25: 254-264Crossref PubMed Scopus (382) Google Scholar, Zhan et al., 2016Zhan T. Rindtorff N. Boutros M. Wnt signaling in cancer.Oncogene. 2016; 36: 1461Crossref PubMed Scopus (1400) Google Scholar). Wnt ligands bind to LRP5/6 and Frizzled family transmembrane receptors, which triggers a signaling cascade that inhibits GSK3 to stabilize β-catenin. This transcriptional activator then translocates to the nucleus, where it binds to the lymphoid enhancer factor/T cell factor (LEF/TCF) and activates Wnt target genes (MacDonald and He, 2012MacDonald B.T. He X. Frizzled and LRP5/6 receptors for Wnt/beta-catenin signaling.Cold Spring Harb Perspect. Biol. 2012; 4Crossref PubMed Scopus (382) Google Scholar, Niehrs, 2012Niehrs C. The complex world of WNT receptor signalling.Nat. Rev. Mol. Cell Biol. 2012; 13: 767-779Crossref PubMed Scopus (972) Google Scholar). Wnts can engage various co-receptors and relevant for this study are syndecan transmembrane proteoglycans, which negatively regulate Wnt/β-catenin signaling (Astudillo et al., 2014Astudillo P. Carrasco H. Larrain J. Syndecan-4 inhibits Wnt/beta-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.Int. J. Biochem. Cell Biol. 2014; 46: 103-112Crossref PubMed Scopus (23) Google Scholar). At the receptor level, Wnt signaling is tightly controlled by secreted antagonists such as Cerberus, Frzb/sFRP3, Dkk1, 2, 4, and Wif1, which inhibit either Wnt ligands or their receptors (Cruciat and Niehrs, 2013Cruciat C.M. Niehrs C. Secreted and transmembrane wnt inhibitors and activators.Cold Spring Harb Perspect. Biol. 2013; 5: a015081Crossref PubMed Scopus (423) Google Scholar). For example, the Wnt signaling antagonist Dkk1 binds to LRP5/6 and induces receptor internalization (Mao et al., 2001Mao B. Wu W. Li Y. Hoppe D. Stannek P. Glinka A. Niehrs C. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.Nature. 2001; 411: 321-325Crossref PubMed Scopus (894) Google Scholar, Mao et al., 2002Mao B. Wu W. Davidson G. Marhold J. Li M. Mechler B.M. Delius H. Hoppe D. Stannek P. Walter C. et al.Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling.Nature. 2002; 417: 664-667Crossref PubMed Scopus (859) Google Scholar). Conversely, secreted Wnt agonists of the R-spondin family increase surface levels of Frizzled and LRP6 by preventing Wnt receptor ubiquitination and surface clearance (Binnerts et al., 2007Binnerts M.E. Kim K.A. Bright J.M. Patel S.M. Tran K. Zhou M. Leung J.M. Liu Y. Lomas 3rd, W.E. Dixon M. et al.R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6.Proc. Natl. Acad. Sci. USA. 2007; 104: 14700-14705Crossref PubMed Scopus (222) Google Scholar, Hao et al., 2012Hao H.X. Xie Y. Zhang Y. Charlat O. Oster E. Avello M. Lei H. Mickanin C. Liu D. Ruffner H. et al.ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner.Nature. 2012; 485: 195-200Crossref PubMed Scopus (618) Google Scholar). In this study, we identify Angiopoietin-like 4 (ANGPTL4) as a Wnt inhibitor. ANGPTL4 belongs to a family of secreted glycoproteins consisting of eight members, whose cellular functions and mechanism of action remain incompletely understood (Santulli, 2014Santulli G. Angiopoietin-like proteins: a comprehensive look.Front. Endocrinol. 2014; 5: 4Crossref PubMed Scopus (191) Google Scholar, Zhu et al., 2012Zhu P. Goh Y.Y. Chin H.F. Kersten S. Tan N.S. Angiopoietin-like 4: a decade of research.Biosci. Rep. 2012; 32: 211-219Crossref PubMed Scopus (185) Google Scholar). ANGPTL4 is involved in a variety of biological processes. The best-characterized role of ANGPTL4 is the regulation of lipid metabolism. ANGPTL4 attenuates the clearance of circulating triglycerides by inhibition of lipoprotein lipase (Lei et al., 2011Lei X. Shi F. Basu D. Huq A. Routhier S. Day R. Jin W. Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity.J. Biol. Chem. 2011; 286: 15747-15756Crossref PubMed Scopus (106) Google Scholar, Sukonina et al., 2006Sukonina V. Lookene A. Olivecrona T. Olivecrona G. Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue.Proc. Natl. Acad. Sci. USA. 2006; 103: 17450-17455Crossref PubMed Scopus (314) Google Scholar, Yoshida et al., 2002Yoshida K. Shimizugawa T. Ono M. Furukawa H. Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase.J. Lipid Res. 2002; 43: 1770-1772Crossref PubMed Scopus (305) Google Scholar), which makes this cytokine a key effector in, e.g., cardiovascular disease and metabolic disorder, and renders it a potential drug target (Dewey et al., 2016Dewey F.E. Gusarova V. O'Dushlaine C. Gottesman O. Trejos J. Hunt C. Van Hout C.V. Habegger L. Buckler D. Lai K.M. et al.Inactivating variants in ANGPTL4 and risk of coronary artery disease.N. Engl. J. Med. 2016; 374: 1123-1133Crossref PubMed Scopus (310) Google Scholar, Hato et al., 2008Hato T. Tabata M. Oike Y. The role of angiopoietin-like proteins in angiogenesis and metabolism.Trends Cardiovasc. Med. 2008; 18: 6-14Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, Kersten, 2005Kersten S. Regulation of lipid metabolism via angiopoietin-like proteins.Biochem. Soc. Trans. 2005; 33: 1059-1062Crossref PubMed Scopus (110) Google Scholar). Moreover, ANGPTL4 is linked to cancer development and metastasis (Nakayama et al., 2011Nakayama T. Hirakawa H. Shibata K. Nazneen A. Abe K. Nagayasu T. Taguchi T. Expression of angiopoietin-like 4 (ANGPTL4) in human colorectal cancer: ANGPTL4 promotes venous invasion and distant metastasis.Oncol. Rep. 2011; 25: 929-935Crossref PubMed Scopus (69) Google Scholar, Padua et al., 2008Padua D. Zhang X.H. Wang Q. Nadal C. Gerald W.L. Gomis R.R. Massague J. TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4.Cell. 2008; 133: 66-77Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, Tan et al., 2012Tan M.J. Teo Z. Sng M.K. Zhu P. Tan N.S. Emerging roles of angiopoietin-like 4 in human cancer.Mol. Cancer Res. 2012; 10: 677-688Crossref PubMed Scopus (120) Google Scholar), but its molecular function in these disorders is largely unknown. Here, we describe ANGPTL4 as a Wnt signaling antagonist and show that it inhibits canonical Wnt signaling in mammalian cells and in Xenopus embryos. ANGPTL4 binds to syndecans and triggers their endocytosis along with the Wnt co-receptor LRP6, which becomes degraded. In Xenopus embryos, angptl4 is prominently expressed in the Spemann organizer and notochord, where it is required to attenuate Wnt activity to promote notochord development. In a genome-wide small interfering RNA (siRNA) screen for Wnt/β-catenin regulators in HEK293T cells (Cruciat et al., 2010Cruciat C.M. Ohkawara B. Acebron S.P. Karaulanov E. Reinhard C. Ingelfinger D. Boutros M. Niehrs C. Requirement of prorenin receptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling.Science. 2010; 327: 459-463Crossref PubMed Scopus (448) Google Scholar, Glinka et al., 2011Glinka A. Dolde C. Kirsch N. Huang Y.L. Kazanskaya O. Ingelfinger D. Boutros M. Cruciat C.M. Niehrs C. LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and Wnt/PCP signalling.EMBO Rep. 2011; 12: 1055-1061Crossref PubMed Scopus (412) Google Scholar), we identified ANGPTL4 as a candidate negative regulator. In non-small cell lung cancer H1703 cells, knockdown of ANGPTL4 by siRNA activated Wnt signaling in β-catenin/TCF reporter assays (TopFlash), while siANGPTL3 and siANGPTL5 had no effect (Figures 1A, S1A, and S1B). Similarly, siANGPTL4 activated Wnt signaling in HEK293T cells, albeit to a lesser extent, and this effect was blocked by addition of exogenous ANGPTL4, supporting specificity (Figure 1B). Consistent with Wnt reporter activation, siANGPTL4 strongly induced expression of the Wnt target gene AXIN2 (Figure 1C) and augmented β-catenin levels (Figure 1D). Moreover, siANGPTL4 enhanced Wnt reporter assays stimulated either by a combination of Wnt1, Fz8, and LRP6, or by Wnt3a, but not by downstream components of the pathway, such as Disheveled (DVL1) or β-catenin (Figure 1E), suggesting that ANGPTL4 may act at the level of Wnt receptors. Indeed, depletion of ANGPTL4 increased protein levels of the Wnt co-receptor LRP6 (Figure 1F), and the magnitude of Wnt reporter activation by siANGPTL4 correlated with LRP6 protein levels, i.e., it was high in cells with high LRP6 and ANGPTL4 levels (H1703, H1299) and lower in cells with low receptor and ANGPTL4 levels (HEK293T) (Figures S1C–S1I). To investigate the physiological relevance of ANGPTL4-mediated Wnt inhibition, we turned to Xenopus embryos, where Wnt signaling prominently regulates early developmental processes. The Xenopus tropicalis angptl4 homolog was maternally expressed at low levels (Figure S2A, qRT-PCR). Interestingly, data mining revealed that angptl4 transcripts are highly enriched on the dorsal side of Xenopus laevis gastrulae (Figure S2B) and indeed, angptl4 was expressed in the Spemann organizer (Figure 2A). Since ANGPTL4 inhibits Wnt signaling, this expression domain is particularly revealing since the Spemann organizer is the source of multiple Wnt antagonists, e.g., cerberus, dkk1, and sfrp3/frzb (Bouwmeester et al., 1996Bouwmeester T. Kim S. Sasai Y. Lu B. De Robertis E.M. Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann's organizer.Nature. 1996; 382: 595-601Crossref PubMed Scopus (634) Google Scholar, Glinka et al., 1998Glinka A. Wu W. Delius H. Monaghan A.P. Blumenstock C. Niehrs C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction.Nature. 1998; 391: 357-362Crossref PubMed Scopus (1349) Google Scholar, Leyns et al., 1997Leyns L. Bouwmeester T. Kim S.H. Piccolo S. De Robertis E.M. Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.Cell. 1997; 88: 747-756Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). At later stages, angptl4 showed highly differential expression in multiple organs during organogenesis (Figure S2C). However, in neurulae and tailbud embryos, angptl4 expression was largely restricted to the notochord (Figures 2B, 2C, and S2C). To address the physiological role of angptl4, we performed Xenopus gain-of-function and loss-of-function experiments. Wnt signaling plays an important role during early Xenopus development in mesodermal and anterior-posterior neural patterning (Hoppler et al., 1996Hoppler S. Brown J.D. Moon R.T. Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.Genes Dev. 1996; 10: 2805-2817Crossref PubMed Scopus (305) Google Scholar, Hoppler and Moon, 1998Hoppler S. Moon R.T. BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm.Mech. Dev. 1998; 71: 119-129Crossref PubMed Scopus (142) Google Scholar, Kiecker and Niehrs, 2001Kiecker C. Niehrs C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus.Development. 2001; 128: 4189-4201PubMed Google Scholar). Overexpression of Wnt inhibitors in Xenopus embryos typically induces enlarged cement glands and inhibits midbrain development (en2 expression) (Kiecker and Niehrs, 2001Kiecker C. Niehrs C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus.Development. 2001; 128: 4189-4201PubMed Google Scholar, Leyns et al., 1997Leyns L. Bouwmeester T. Kim S.H. Piccolo S. De Robertis E.M. Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.Cell. 1997; 88: 747-756Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). This was also the case when angptl4 mRNA was microinjected, phenocopying lrp6 antisense Morpholino oligonucleotide (Mo) injection (Figures 2D and 2E). For loss-of-function, we designed two non-overlapping splice-site antisense Mos (Mo1, Mo2) targeting angptl4, microinjection of which strongly reduced endogenous angptl4 mRNA levels (Figures S2D and S2E). Injection of angptl4 Mo1 and Mo2 resulted in a similar phenotype, characterized by small heads, shortened bent body axes, and defects in melanocyte and eye pigmentation (Figure 2F). Importantly, co-injection of mouse Angptl4 mRNA rescued this Mo1 phenotype in a dose-dependent manner (Figure 2F). Moreover, co-injection of lrp6 Mo also rescued the angptl4 Mo1 phenotype (Figure 2G), indicating that the malformations are due to elevated Wnt signaling. There are two phases of Wnt signaling in early Xenopus embryos, pre- and post-midblastula transition (MBT), which have the opposite effect on Spemann organizer function. Experimentally, both phases can be distinguished by injection of Wnt RNA versus Wnt DNA, which is expressed only after MBT (Christian and Moon, 1993Christian J.L. Moon R.T. Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.Genes Dev. 1993; 7: 13-28Crossref PubMed Scopus (379) Google Scholar). Pre-MBT Wnt signaling induces the Spemann organizer and marker genes such as Xnr3. Post-MBT Wnt signaling inhibits Spemann organizer and its derivatives (Christian and Moon, 1993Christian J.L. Moon R.T. Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.Genes Dev. 1993; 7: 13-28Crossref PubMed Scopus (379) Google Scholar, Hoppler et al., 1996Hoppler S. Brown J.D. Moon R.T. Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.Genes Dev. 1996; 10: 2805-2817Crossref PubMed Scopus (305) Google Scholar, Hoppler and Moon, 1998Hoppler S. Moon R.T. BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm.Mech. Dev. 1998; 71: 119-129Crossref PubMed Scopus (142) Google Scholar). Angptl4 Mo1 enhanced the expression of the Wnt target gene Xnr3, a marker for Nieuwkoop center, pre-MBT Wnt signaling. This effect was rescued by co-injection of mouse Angptl4 mRNA as well (Figure 2H), further corroborating Mo specificity and the inhibitory role of Angptl4 in Wnt signaling. Notochord formation requires inhibition of post-MBT Wnt signaling to prevent mesodermal precursors from undergoing muscle differentiation (Hoppler et al., 1996Hoppler S. Brown J.D. Moon R.T. Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.Genes Dev. 1996; 10: 2805-2817Crossref PubMed Scopus (305) Google Scholar, Hoppler and Moon, 1998Hoppler S. Moon R.T. BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm.Mech. Dev. 1998; 71: 119-129Crossref PubMed Scopus (142) Google Scholar). Thus, if Angptl4 is a bona fide Wnt inhibitor acting in notochord, its loss-of-function should result in a switch from notochord to muscle fate. Indeed, depletion of angptl4 with either Morpholino reduced the notochord marker chordin, as assessed by whole-mount in situ hybridization at early neurula stage, phenocopying Wnt8 DNA gain of function (Figure 3A). Moreover, in animal cap explants induced to form notochord by activin treatment, depletion of angptl4 with either Morpholino reduced the expression of the notochord/dorsal mesodermal markers noggin and goosecoid (gsc), while inducing the muscle marker myf5, similar to Wnt8 overexpression (Figures 3B and 3C). Corroborating these findings, angptl4 Mo1 increased myf5 expression in gastrulae and neurulae, as did Wnt8 gain-of-function (Figures 3D and 3E, quantified in Figures S3A and S3B). In line with this, angptl4 mRNA increased the notochord marker Xnot2 (Figure 3F, quantified in Figure S3C), whereas the muscle marker myf5 was decreased (Figure 3G, quantified in Figure S3D). This effect was rescued by activation of the Wnt signaling pathway via co-injection of Wnt8 DNA (Figures 3F and 3G, quantified in Figures S3C and S3D). Taken together, these results support a model in which Xenopus Angptl4 acts as a spatially restricted Wnt antagonist that maintains notochord identity by preventing muscle differentiation. To explore the molecular mechanism by which ANGPTL4 inhibits Wnt signaling, we first determined whether known angiopoietin-like protein interactors are Wnt regulators. Leukocyte immunoglobulin-like receptor B2 (LILRB2) serves as a receptor for ANGPTL2 and ANGPTL5, and it was suggested that it may also bind other ANGPTL proteins (Zheng et al., 2012Zheng J. Umikawa M. Cui C. Li J. Chen X. Zhang C. Huynh H. Kang X. Silvany R. Wan X. et al.Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development.Nature. 2012; 485: 656-660Crossref PubMed Scopus (183) Google Scholar); however, we did not observe binding of ANGPTL4 to LILRB2 (Figure S4A), and siLILRB2 had no effect on Wnt signaling (Figure S4B). Likewise, Wnt signaling was not affected by depletion of lipoprotein lipase (LPL) (Figure S4B), which is bound and inhibited by ANGPTL4 (Yoshida et al., 2002Yoshida K. Shimizugawa T. Ono M. Furukawa H. Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase.J. Lipid Res. 2002; 43: 1770-1772Crossref PubMed Scopus (305) Google Scholar). While our results indicated that ANGPTL4 functions in Wnt signaling at the receptor level (Figures 1E and 1F), in vitro binding assays failed to support direct binding to either Frizzled or LRP6 (data not shown and see below). In summary, these results suggest that ANGPTL4 controls Wnt signaling via an as yet unknown interaction partner. We screened for novel ANGPTL4 interactors in HepG2 cells by mass spectrometry following pull-down of soluble ANGPTL4 (Table S1). Intriguingly, among the most prominent hits were syndecans (SDC) 1, 2, and 4. Syndecans are transmembrane proteins that act as co-receptors in various signaling pathways (Tkachenko et al., 2005Tkachenko E. Rhodes J.M. Simons M. Syndecans: new kids on the signaling block.Circ. Res. 2005; 96: 488-500Crossref PubMed Scopus (363) Google Scholar). In the context of Wnt signaling, syndecans activate the Wnt/PCP pathway (Munoz et al., 2006Munoz R. Moreno M. Oliva C. Orbenes C. Larrain J. Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos.Nat. Cell Biol. 2006; 8: 492-500Crossref PubMed Scopus (116) Google Scholar, Ohkawara et al., 2011Ohkawara B. Glinka A. Niehrs C. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.Dev. Cell. 2011; 20: 303-314Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and inhibit Wnt/β-catenin signaling by reducing LRP6 levels (Astudillo et al., 2014Astudillo P. Carrasco H. Larrain J. Syndecan-4 inhibits Wnt/beta-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.Int. J. Biochem. Cell Biol. 2014; 46: 103-112Crossref PubMed Scopus (23) Google Scholar). Syndecans can also regulate FGF signaling (Elfenbein and Simons, 2013Elfenbein A. Simons M. Syndecan-4 signaling at a glance.J. Cell Sci. 2013; 126: 3799-3804Crossref PubMed Scopus (143) Google Scholar), but siANGPTL4 had no effect on FGF8 reporter assays (Figure S4C). We confirmed binding of ANGPTL4 to SDC in cell-surface binding assays, which showed that soluble ANGPTL4 specifically binds all four syndecan family members (SDC1–4) (Figure 4A). Moreover, in ELISA, alkaline phosphatase (AP)-tagged soluble SDC4 bound dose dependently to immobilized ANGPTL4 (Figure 4B). A characteristic feature of syndecans is their ability to induce endocytosis upon ligand binding (Fuki et al., 1997Fuki I.V. Kuhn K.M. Lomazov I.R. Rothman V.L. Tuszynski G.P. Iozzo R.V. Swenson T.L. Fisher E.A. Williams K.J. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro.J. Clin. Invest. 1997; 100: 1611-1622Crossref PubMed Scopus (206) Google Scholar, Tkachenko and Simons, 2002Tkachenko E. Simons M. Clustering induces redistribution of syndecan-4 core protein into raft membrane domains.J. Biol. Chem. 2002; 277: 19946-19951Crossref PubMed Scopus (97) Google Scholar). We therefore investigated whether ANGPTL4 undergoes SDC-dependent internalization (Figures 4C, S4D, and S4E). Incubation of HepG2 cells with horseradish peroxidase (HRP)-tagged ANGPTL4 at 37°C led to its internalization within 40 min (Figures 4C and S4E). In contrast, no internalization was observed when cells were incubated on ice. Treatment of cells with chlorate to inhibit sulfation of SDC heparan sulfate chains almost completely abrogated ANGPTL4 endocytosis, suggesting that heparan sulfate proteoglycans are required for ANGPTL4 internalization. Simultaneous depletion of all four syndecans using siRNA blocked ANGPTL4 internalization, while depletion of any single syndecan had no effect, indicating that syndecans act redundantly in ANGPTL4 internalization. To determine the endocytic route, cells were treated with inhibitors of clathrin- or caveolin-mediated endocytosis (Figure S4D). The clathrin inhibitor monodansylcadaverine (MDC) blocked ANGPTL4 internalization, unlike inhibitors of caveolin-mediated endocytosis, nystatin and filipin. Concordantly, depletion of clathrin by siRNA reduced ANGPTL4 cytoplasmic translocation, whereas siCaveolin had no effect (Figure S4D). ANGPTL4 is rapidly cleaved upon secretion, and its N- and C-terminal fragments (nANGPTL4 and cANGPTL4, respectively) exert discrete biological functions (Ge et al., 2004Ge H. Yang G. Huang L. Motola D.L. Pourbahrami T. Li C. Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4.J. Biol. Chem. 2004; 279: 2038-2045Crossref PubMed Scopus (124) Google Scholar, Zhu et al., 2012Zhu P. Goh Y.Y. Chin H.F. Kersten S. Tan N.S. Angiopoietin-like 4: a decade of research.Biosci. Rep. 2012; 32: 211-219Crossref PubMed Scopus (185) Google Scholar). Whereas nANGPTL4 is an orphan ligand that mainly controls lipid metabolism, cANGPTL4 binds, e.g., β-integrins, VE-cadherin, and claudin-5 to induce vascular leakiness and tumor progression (Huang et al., 2011Huang R.L. Teo Z. Chong H.C. Zhu P. Tan M.J. Tan C.K. Lam C.R. Sng M.K. Leong D.T. Tan S.M. et al.ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters.Blood. 2011; 118: 3990-4002Crossref PubMed Scopus (167) Google Scholar, Zhu et al., 2011Zhu P. Tan M.J. Huang R.L. Tan C.K. Chong H.C. Pal M. Lam C.R. Boukamp P. Pan J.Y. Tan S.H. et al.Angiopoietin-like 4 protein elevates the prosurvival intracellular O2(-):H2O2 ratio and confers anoikis resistance to tumors.Cancer Cell. 2011; 19: 401-415Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). We observed that nANGPTL4, but not cANGPTL4, was rapidly internalized in HepG2 cells (Figures 4D and S4F). Likewise, in vitro binding assays showed that SDC4 specifically binds nANGPTL4 (Figure 4E). The results corroborate that syndecans bind and internalize nANGPTL4 by clathrin-mediated endocytosis. In Wnt reporter assays, siSDC1 and siANGPTL4 synergistically activated β-catenin-dependent gene transcription (Figure 4F), indicating that the proteins interact not only biochemically but also functionally. In Xenopus explants, injection of dominant-negative SDC4 (SDC4ΔC and SDC4ΔTMC; Ohkawara et al., 2011Ohkawara B. Glinka A. Niehrs C. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.Dev. Cell. 2011; 20: 303-314Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) reduced the expression of the dorsal mesodermal marker goosecoid (gsc), whereas the muscle marker myf5 was induced (Figures 4G and S4G). This phenocopies angptl4 Mo injection (Figures 3B and 3C) and suggests that ANGPTL4 and syndecans act in the same pathway. Indeed, co-injection of angptl4 Mo and sdc4 Mo synergized in reducing notochord/dorsal mesodermal markers noggin and goosecoid (gsc), while synergistically inducing the muscle marker myf5 (Figures 4H and S4H). This confirms the effects observed in vitro and indicates functional interaction of Sdc4 and Angptl4 in Xenopus during notochord formation. Although we were unable to detect direct binding of ANGPTL4 to LRP6 (Figure 4A), the observation that both syndecans (Astudillo et al., 2014Astudillo P. Carrasco H. Larrain J. Syndecan-4 inhibits Wnt/beta-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.Int. J. Biochem. Cell Biol. 2014; 46: 103-112Crossref PubMed Scopus (23) Google Scholar) and ANGPTL4 negatively regulate LRP6 levels raised the possibility that ANGPTL4 controls Wnt signaling by recruiting syndecans to LRP6. To determine if LRP6 and LRP5 are required for the effect of ANGPTL4 on Wnt signaling, we generated LRP6 single and LRP5/6 double knockout HEK293T (HEK293TΔLRP5/6) cells by CRISPR/Cas9-mediated gene editing (Figure S5A). These cells lack LRP5/6 proteins and are unresponsive to Wnt3a; however they retain the ability to upregulate Wnt signaling upon siAPC treatment, consistent with a block of Wnt signaling at the receptor level (Figures S5A and S5B). Importantly, in these HEK293TΔLRP5/6 cells, siANGPTL4 depletion failed to induce Wnt reporter activity (Figure 5A). This effect was restored by re-expression of LRP6 in HEK293TΔLRP6 cells (Figure S5C). Conversely, LRP6 overexpression strongly synergized with siANGPTL4 in Wnt reporter induction (Figure 5B). These results confirm the requirement of LRP5/6 for the inhibitory effect of ANGPTL4 on Wnt/β-catenin signaling. To investigate if ANGPTL4 promotes the association of SDC4 and LRP6, we made use of the proximity ligation assay (PLA). Addition of nANGPTL4 conditioned medium increased the interaction of LRP6 and SDC4, as indicated by the presence of more and larger SDC4-LRP6 clusters in nANGPTL4-treated cells, whereas cANGPTL4 had no effect (Figures 5C, 5D, S5D, and S5E). This LRP6-SDC4 interaction induced by ANGPTL4 was sensitive to chlorate treatment (Figure S5F), suggesting that binding of ANGPTL4 to syndecans is required for LRP6-SDC4 clustering. In Xenopus embryos, angptl4 mRNA reduced LRP6 protein levels, and this effect was reversed by syndecan 4 Mo (Figure 5E). In cell-surface biotinylation assays, siANGPTL4 increased cell-surface LRP6 without affecting transferrin receptor levels (Figure 5F). Moreover, nANGPTL4 co-localized with LRP6 in endo-/lysosomal vesicles when lysosomal degradation was inhibited with bafilomycin (Figure 5G) or chloroquine (not shown). Taken together, the results support a model in which ANGPTL4 interaction with syndecans promotes the formation of a ternary complex with LRP6, resulting in Wnt receptor clearance and lysosomal degradation. Live-cell imaging of SDC4-LRP6 interaction using a split-fluorescent protein-based bimolecular fluorescence complementation (BIFC) assay (Kodama and Hu, 2010Kodama Y. Hu C.D. An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio.BioTechniques. 2010; 49: 793-805Crossref PubMed Scopus (138) Google Scholar) corroborated this model (Figures 5H and 5I): in untreated cells, addition of nANGPTL4 induced a marked decay of the LRP6-SDC4 BIFC signal (Figure 5H); In contrast, when lysosomal degradation was inhibited using chloroquine, nANGPTL4 strongly increased the BIFC signal (Figure 5I). A control BIFC signal using the fibronectin leucine-rich transmembr" @default.
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• W2763150312 date "2017-10-01" @default.
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• W2763150312 title "Angiopoietin-like 4 Is a Wnt Signaling Antagonist that Promotes LRP6 Turnover" @default.
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• W2763150312 doi "https://doi.org/10.1016/j.devcel.2017.09.011" @default.
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