Abstract
LDL receptor–related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical β-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced β-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-β-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving β-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.
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Abbreviations
- LRPs:
-
LDL receptor–related proteins
- tPA:
-
Tissue plasminogen activator
- MβCD:
-
Methyl-β-cyclodextrin
- mAb:
-
Monoclonal antibody
- SDS-PAGE:
-
Sodium-dodecyl sulfate polyacrylamide gel electrophoresis
- PVDF:
-
Polyvinylidene difluoride
- HRP:
-
Horseradish peroxidase
References
Brown DA, London E (1998) Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14:111–136. https://doi.org/10.1146/annurev.cellbio.14.1.111
Brown DA, London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275:17221–17224. https://doi.org/10.1074/jbc.R000005200
Díaz M, Fabelo N, Ferrer I, Marín R (2018) "Lipid raft aging" in the human frontal cortex during nonpathological aging: gender influences and potential implications in Alzheimer's disease. Neurobiol Aging 67:42–52. https://doi.org/10.1016/j.neurobiolaging.2018.02.022
Garofalo T, Misasi R, Mattei V, Giammarioli AM, Malorni W, Pontieri GM, Pavan A, Sorice M (2003) Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas. Evidence for caspase-8-ganglioside interaction in T cells. J Biol Chem 278:8309–8315 http://www.jbc.org/content/278/10/8309
Garofalo T, Giammarioli AM, Misasi R, Tinari A, Manganelli V, Gambardella L, Pavan A, Malorni W, Sorice M (2005) Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells. Cell Death Differ 12(11):1378–1389. https://doi.org/10.1038/sj.cdd.4401672
Garofalo T, Ferri A, Sorice M, Azmoon P, Grasso M, Mattei V, Capozzi A, Manganelli V, Misasi R (2018) Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells. Mol Cell Neurosci 88:167–176. https://doi.org/10.1016/j.mcn.2018.01.007
Goel S, Chin EN, Fakhraldeen SA, Berry SM, Beebe DJ, Alexander CM (2012) Both LRP5 and LRP6 receptors are required to respond to physiological Wnt ligands in mammary epithelial cells and fibroblasts. J Biol Chem 287:16454–16466. https://doi.org/10.1074/jbc.M112.362137
Haack F, Lemcke H, Ewald R, Rharass T, Uhrmacher AM (2015) Spatio-temporal model of endogenous ROS and Raft-dependent WNT/Beta-catenin signaling driving cell fate commitment in human neural progenitor cells. PLoS Comput Biol 11(3):e1004106. https://doi.org/10.1371/journal.pcbi.1004106
He X, Semenov M, Tamai K, Zeng X (2004) LDL receptor-related proteins 5 and 6 in Wnt/β-catenin signaling: arrows point the way. Development 131:1663–1677. https://doi.org/10.1242/dev.01117
Herz J, Strickland DK (2001) LRP: a multifunctional scavenger and signaling receptor. J Clin Invest 108:779–784. https://doi.org/10.1172/JCI200113992
Huelsken J, Behrens J (2002) The Wnt signalling pathway. J Cell Sci 115:3977–3978. https://doi.org/10.1242/jcs.00089
Lara-Castillo N, Johnson ML (2015) LRP receptor family member associated bone disease. N Rev Endocr Metab Disord 16:141–148. https://doi.org/10.1007/s11154-015-9315-2
Laudati E, Gilder AS, Lam MS, Misasi R, Sorice M, Gonias SL, Mantuano E (2016) The activities of LDL receptor-related Protein-1 (LRP1) compartmentalize into distinct plasma membrane microdomains. Mol Cell Neurosci 76:42–51. https://doi.org/10.1016/j.mcn.2016.08.006
Lee SH, Ko HM, Kwon KJ, Lee J, Han SH, Han DW, Cheong JH, Ryu JH, Shin CY (2014) tPA regulates Neurite outgrowth by phosphorylation of LRP5/6 in neural progenitor cells. Mol Neurobiol 49:199–215. https://doi.org/10.1007/s12035-013-8511-x
Malorni W, Giammarioli A, Garofalo T, Sorice M (2007) Dynamics of lipid raft components during lymphocyte apoptosis: the paradigmatic role of GD3. Apoptosis 12:941–949. https://doi.org/10.1007/s10495-007-0757-1
Mantuano E, Brifault C, Lam MS, Azmoon P, Gilder AS, Gonias SL (2016) LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response. Proc Natl Acad Sci U S A 113:1369–1374. https://doi.org/10.1073/pnas.1515480113
Mao X, Tey SK, Ko FCF, Kwong EML, Gao Y, Ng IO, Cheung ST, Guan XY, Yam JWP (2019) C-terminal truncated HBx protein activates caveolin-1/LRP6/β-catenin/FRMD5 q1axis in promoting hepatocarcinogenesis. Cancer Lett 444:60–69. https://doi.org/10.1016/j.canlet.2018.12.003
Mattei V, Manganelli V, Martellucci S, Capozzi A, Mantuano E, Longo A, Ferri A, Garofalo T, Sorice M, Misasi R (2019) A multimolecular signaling complex including PrPC and LRP1 is strictly dependent on lipid rafts and is essential for the function of tissue plasminogen activator. J Neurochem. https://doi.org/10.1111/jnc.14891
Mollinedo F, Gajate C (2015) Lipid rafts as major platforms for signaling regulation in cancer. Adv Biol Regul 57:130–146. https://doi.org/10.1016/j.jbior.2014.10.003
Nag JK, Kancharla A, Maoz M, Turm H, Agranovich D, Gupta CL, Uziely B, Bar-Shavit R (2017) Low-density lipoprotein receptor-related protein 6 is a novel coreceptor of protease-activated receptor-2 in the dynamics of cancer-associated β-catenin stabilization. Oncotarget 8:38650–38667. https://doi.org/10.18632/oncotarget.16246
Özhan G, Sezgin E, Wehner D, Pfister AS, Kühl SJ, Kagermeier-Schenk B, Kühl M, Schwille P, Weidinger G (2013) Lypd6 enhances Wnt/β-catenin signaling by promoting Lrp6 phosphorylation in raft plasma membrane domains. Dev Cell 26:331–345. https://doi.org/10.1016/j.devcel.2013.07.020
Ring L, Neth P, Weber C, Steffens S, Faussner A (2014) β-Catenin-dependent pathway activation by both promiscuous “canonical” WNT3a–, and specific “noncanonical” WNT4– and WNT5a–FZD receptor combinations with strong differences in LRP5 and LRP6 dependency. Cell Signal 26:260–267. https://doi.org/10.1016/j.cellsig.2013.11.021
Sakane H, Yamamoto H, Kikuchi A (2010) Lrp6 is internalized by dkk1 to suppress its phosphorylation in the lipid raft and is recycled for reuse. J Cell Sci 123:360–368. https://doi.org/10.1242/jcs.058008
Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39. https://doi.org/10.1038/35036052
Song Z, Zhu T, Zhou X, Barrow P, Yang W, Cui Y, Yang L, Zhao D (2016) Rest alleviates neurotoxic prion peptide-induced synaptic abnormalities, neurofibrillary degeneration and neuronal death partially via LRP6-mediated Wnt-β-catenin signaling. Oncotarget 7:12035–12052. https://doi.org/10.18632/oncotarget.7640
Sorice M, Garofalo T, Misasi R, Manganelli V, Vona R, Malorni W (2012a) Ganglioside GD3 as a raft component in cell death regulation. Anti Cancer Agents Med Chem 12:376–382. https://doi.org/10.2174/187152012800228670
Sorice M, Mattei V, Matarrese P, Garofalo T, Tinari A, Gambardella L, Ciarlo L, Manganelli V, Tasciotti V, Misasi R, Malorni W (2012b) Dynamics of mitochondrial raft-like microdomains in cell life and death. Commun Integr Biol 5:217–219. https://doi.org/10.4161/cib.19145
Tao J, Calvisi DF, Ranganathan S, Cigliano A, Zhou L, Singh S, Jiang L, Fan B, Terracciano L, Armeanu-Ebinger S, Ribback S, Dombrowski F, Evert M, Chen X, Monga SPS (2014) Activation of β catenin and Yap1 in human Hepatoblastoma and induction of Hepatocarcinogenesis in mice. Gastroenterology 147:690–701. https://doi.org/10.1053/j.gastro.2014.05.004
Whitehead SN, Gangaraju S, Aylsworth A, Hou ST (2012) Membrane raft disruption results in neuritic retraction prior to neuronal death in cortical neurons. Bioscience Trends 6:183–191. https://doi.org/10.5582/bst.2012.v6.4.183
Yamamoto H, Sakane H, Yamamoto H, Michiue T, Kikuchi A (2008) Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling. Dev Cell 15:37–48. https://doi.org/10.1016/j.devcel.2008.04.015
Yao Q, An Y, Hou W, Cao YN, Yao MF, Ma NN, Hou L, Zhang H, Liu HJ, Zhang B (2017) LRP6 promotes invasion and metastasis of colorectal cancer through cytoskeleton dynamics. Oncotarget 8:109632–109645. https://doi.org/10.18632/oncotarget.22759
Zilberberg A, Yaniv A, Gazit A (2004) The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway. J Biol Chem 279:17535–17542. https://doi.org/10.1074/jbc.M311292200
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This research did not receive any specific grant from funding agencies in the public, commercial, and not-for-profit sectors.
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Gloria Riitano, Valeria Manganelli, Antonella Capozzi, Vincenzo Mattei, Serena Recalchi, and Stefano Martellucci gave substantial contributions to the acquisition, analysis and interpretation of data; Agostina Longo, Roberta Misasi, Tina Garofalo and Maurizio Sorice gave substantial contributions to the conception and design of the work.
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Riitano, G., Manganelli, V., Capozzi, A. et al. LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells. J. Cell Commun. Signal. 14, 315–323 (2020). https://doi.org/10.1007/s12079-020-00551-w
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DOI: https://doi.org/10.1007/s12079-020-00551-w