The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles
The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrPC). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrPC. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrPC-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrPC signaling.
KeywordsExosomes Chaperones Prion protein STI1 Extracellular vesicles
We are very grateful to Dr. Wes Sundquist for the donation of VPS4A plasmids. We thank Leica Microsystems and Ms. Lianne Dale for help in experiments done in a demo Leica GSD unit. This investigation was supported by grants from FAPESP to VRM (2009/14027-2) and GNMH (2012/04370-4), PrioNet-Canada, Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation and Ontario Research Fund to MAMP, CNPq and FAPERJ to RL, FAPESP fellowships to CA, MVSD, MR and BS, and a postdoctoral CNPq fellowship to IPC are gratefully acknowledged.
Conflict of interest
The authors declare that there are no conflicts of interest.
- 11.Wang TH, Chao A, Tsai CL, Chang CL, Chen SH, Lee YS, Chen JK, Lin YJ, Chang PY, Wang CJ, Chao AS, Chang SD, Chang TC, Lai CH, Wang HS (2010) Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation. Mol Cell Proteomics 9:1873–1884. doi: 10.1074/mcp.M110.000802 PubMedCrossRefGoogle Scholar
- 12.Zanata SM, Lopes MH, Mercadante AF, Hajj GN, Chiarini LB, Nomizo R, Freitas AR, Cabral AL, Lee KS, Juliano MA, de OE, Jachieri SG, Burlingame A, Huang L, Linden R, Brentani RR, Martins VR (2002) Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. EMBO J 21:3307–3316. doi: 10.1093/emboj/cdf325 PubMedCrossRefGoogle Scholar
- 14.Hajj GN, Santos TG, Landenberger MC, Lopes MH (2012) Transmissible spongiform encephalopathies. In: Quevedo A (ed) Brain damage—bridging between basic research and clinics. InTechOpen, Rijeka, pp 197–220Google Scholar
- 15.Roffe M, Beraldo FH, Bester R, Nunziante M, Bach C, Mancini G, Gilch S, Vorberg I, Castilho BA, Martins VR, Hajj GN (2010) Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR. Proc Natl Acad Sci USA 107:13147–13152. doi: 10.1073/pnas.1000784107 PubMedCrossRefGoogle Scholar
- 16.Lopes MH, Hajj GN, Muras AG, Mancini GL, Castro RM, Ribeiro KC, Brentani RR, Linden R, Martins VR (2005) Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways. J Neurosci 25:11330–11339. doi: 10.1523/JNEUROSCI.2313-05.2005 PubMedCrossRefGoogle Scholar
- 17.Beraldo FH, Arantes CP, Santos TG, Queiroz NG, Young K, Rylett RJ, Markus RP, Prado MA, Martins VR (2010) Role of alpha7 nicotinic acetylcholine receptor in calcium signaling induced by prion protein interaction with stress-inducible protein 1. J Biol Chem 285:36542–36550. doi: 10.1074/jbc.M110.157263 PubMedCrossRefGoogle Scholar
- 19.Tsai CL, Tsai CN, Lin CY, Chen HW, Lee YS, Chao A, Wang TH, Wang HS, Lai CH (2012) Secreted stress-induced phosphoprotein 1 activates the ALK2-SMAD signaling pathways and promotes cell proliferation of ovarian cancer cells. Cell Rep 2:283–293. doi: 10.1016/j.celrep.2012.07.002 PubMedCrossRefGoogle Scholar
- 22.Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP, Boireau W, Rouleau A, Simon B, Lanneau D, De TA, Multhoff G, Hamman A, Martin F, Chauffert B, Solary E, Zitvogel L, Garrido C, Ryffel B, Borg C, Apetoh L, Rebe C, Ghiringhelli F (2010) Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest 120:457–471. doi: 10.1172/JCI40483 PubMedGoogle Scholar
- 43.Caetano FA, Lopes MH, Hajj GN, Machado CF, Pinto AC, Magalhaes AC, Vieira MP, Americo TA, Massensini AR, Priola SA, Vorberg I, Gomez MV, Linden R, Prado VF, Martins VR, Prado MA (2008) Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1. J Neurosci 28:6691–6702. doi: 10.1523/JNEUROSCI.1701-08.2008 PubMedCrossRefGoogle Scholar
- 54.Lu H, Daugherty A (2009) Atherosclerosis: cell biology and lipoproteins. Curr Opin Lipidol 20:528–529. doi: 10.1097/MOL.0b013e328332c3bc;00041433-200912000-00014 PubMedCrossRefGoogle Scholar
- 63.Luo X, Zuo X, Zhang B, Song L, Wei X, Zhou Y, Xiao X (2008) Release of heat shock protein 70 and the effects of extracellular heat shock protein 70 on the production of IL-10 in fibroblast-like synoviocytes. Cell Stress Chaperones 13:365–373. doi: 10.1007/s12192-008-0036-2 PubMedCrossRefGoogle Scholar
- 74.Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. (Chapter 3, Unit 3.22). doi: 10.1002/0471143030.cb0322s30
- 76.De MA (2011) Extracellular heat shock proteins, cellular export vesicles, and the stress observation system: a form of communication during injury, infection, and cell damage. It is never known how far a controversial finding will go! Dedicated to Ferruccio Ritossa. Cell Stress Chaperones 16:235–249. doi: 10.1007/s12192-010-0236-4 CrossRefGoogle Scholar
- 77.Faca VM, Ventura AP, Fitzgibbon MP, Pereira-Faca SR, Pitteri SJ, Green AE, Ireton RC, Zhang Q, Wang H, O’Briant KC, Drescher CW, Schummer M, McIntosh MW, Knudsen BS, Hanash SM (2008) Proteomic analysis of ovarian cancer cells reveals dynamic processes of protein secretion and shedding of extra-cellular domains. PLoS ONE 3:e2425. doi: 10.1371/journal.pone.0002425 PubMedCrossRefGoogle Scholar
- 82.Vega VL, Rodriguez-Silva M, Frey T, Gehrmann M, Diaz JC, Steinem C, Multhoff G, Arispe N, De MA (2008) Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J Immunol 180:4299–4307 (pii: 180/6/4299)PubMedGoogle Scholar