UNLABELLED: Liver fibrosis is characterized by the activation and migration of hepatic stellate cells (HSCs), followed by matrix deposition. Recently, several studies have shown the importance of extracellular vesicles (EVs) derived from liver cells, such as hepatocytes and endothelial cells, in liver pathobiology. While most of the studies describe how liver cells modulate HSC behavior, an important gap exists in the understanding of HSC-derived signals and more specifically HSC-derived EVs in liver fibrosis. Here, we investigated the molecules released through HSC-derived EVs, the mechanism of their release, and the role of these EVs in fibrosis. Mass spectrometric analysis showed that platelet-derived growth factor (PDGF) receptor-alpha (PDGFRα) was enriched in EVs derived from PDGF-BB-treated HSCs. Moreover, patients with liver fibrosis had increased PDGFRα levels in serum EVs compared to healthy individuals. Mechanistically, in vitro tyrosine720-to-phenylalanine mutation on the PDGFRα sequence abolished enrichment of PDGFRα in EVs and redirected the receptor toward degradation. Congruently, the inhibition of Src homology 2 domain tyrosine phosphatase 2, the regulatory binding partner of phosphorylated tyrosine720, also inhibited PDGFRα enrichment in EVs. EVs derived from PDGFRα-overexpressing cells promoted in vitro HSC migration and in vivo liver fibrosis. Finally, administration of Src homology 2 domain tyrosine phosphatase 2inhibitor, SHP099, to carbon tetrachloride-administered mice inhibited PDGFRα enrichment in serum EVs and reduced liver fibrosis. CONCLUSION: PDGFRα is enriched in EVs derived from PDGF-BB-treated HSCs in an Src homology 2 domain tyrosine phosphatase 2-dependent manner and these PDGFRα-enriched EVs participate in development of liver fibrosis. (Hepatology 2018;68:333-348).

Department of Pharmacology Charles University Hradec Kralove Czech Republic

Division of Gastroenterology and Hepatology Mayo Clinic Rochester MN

Zobrazit více v PubMed

Kikuchi A, Monga SP. PDGFRalpha in liver pathophysiology: emerging roles in development, regeneration, fibrosis, and cancer. Gene Expr. 2015;16:109–127. PubMed PMC

Mokdad AA, Lopez AD, Shahraz S, Lozano R, Mokdad AH, Stanaway J, Murray CJ, et al. Liver cirrhosis mortality in 187 countries between 1980 and 2010: a systematic analysis. BMC Med. 2014;12:145. PubMed PMC

Shah VH, Bruix J. Antiangiogenic therapy: not just for cancer anymore? Hepatology. 2009;49:1066–1068. PubMed

Kostallari E, Shah VH. Angiocrine signaling in the hepatic sinusoids in health and disease. Am J Physiol Gastrointest Liver Physiol. 2016;311:G246–251. PubMed PMC

Nagy LE. The Role of Innate Immunity in Alcoholic Liver Disease. Alcohol Res. 2015;37:237–250. PubMed PMC

Torok NJ. Dysregulation of redox pathways in liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 2016;311:G667–G674. PubMed PMC

Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol. 2017;14:397–411. PubMed

Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990;61:203–212. PubMed

Gelderloos JA, Rosenkranz S, Bazenet C, Kazlauskas A. A role for Src in signal relay by the platelet-derived growth factor alpha receptor. J Biol Chem. 1998;273:5908–5915. PubMed

Bazenet CE, Gelderloos JA, Kazlauskas A. Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation. Mol Cell Biol. 1996;16:6926–6936. PubMed PMC

Hayes BJ, Riehle KJ, Shimizu-Albergine M, Bauer RL, Hudkins KL, Johansson F, Yeh MM, et al. Activation of platelet-derived growth factor receptor alpha contributes to liver fibrosis. PLoS One. 2014;9:e92925. PubMed PMC

Hirsova P, Ibrahim SH, Krishnan A, Verma VK, Bronk SF, Werneburg NW, Charlton MR, et al. Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes. Gastroenterology. 2016;150:956–967. PubMed PMC

Hirsova P, Ibrahim SH, Verma VK, Morton LA, Shah VH, LaRusso NF, Gores GJ, et al. Extracellular vesicles in liver pathobiology: Small particles with big impact. Hepatology. 2016;64:2219–2233. PubMed PMC

Sato K, Meng F, Glaser S, Alpini G. Exosomes in liver pathology. J Hepatol. 2016;65:213–221. PubMed PMC

Bala S, Petrasek J, Mundkur S, Catalano D, Levin I, Ward J, Alao H, et al. Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases. Hepatology. 2012;56:1946–1957. PubMed PMC

Ibrahim SH, Hirsova P, Tomita K, Bronk SF, Werneburg NW, Harrison SA, Goodfellow VS, et al. Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes. Hepatology. 2016;63:731–744. PubMed PMC

Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, Messer K, et al. Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease. PLoS One. 2014;9:e113651. PubMed PMC

Verma VK, Li H, Wang R, Hirsova P, Mushref M, Liu Y, Cao S, et al. Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles. J Hepatol. 2016;64:651–660. PubMed PMC

Wang R, Ding Q, Yaqoob U, de Assuncao TM, Verma VK, Hirsova P, Cao S, et al. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration. J Biol Chem. 2015;290:30684–30696. PubMed PMC

Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;Chapter 3(Unit 3):22. PubMed

Maiers JL, Kostallari E, Mushref M, deAssuncao TM, Li H, Jalan-Sakrikar N, Huebert RC, et al. The unfolded protein response mediates fibrogenesis and collagen I secretion through regulating TANGO1 in mice. Hepatology. 2017;65:983–998. PubMed PMC

Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Extracellular Vesicles from Bone Marrow-Derived Mesenchymal Stem Cells Improve Survival from Lethal Hepatic Failure in Mice. Stem Cells Transl Med. 2017;6:1262–1272. PubMed PMC

Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, Dingli F, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A. 2016;113:E968–977. PubMed PMC

Haglund K, Sigismund S, Polo S, Szymkiewicz I, Di Fiore PP, Dikic I. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cell Biol. 2003;5:461–466. PubMed

Tallquist M, Kazlauskas A. PDGF signaling in cells and mice. Cytokine Growth Factor Rev. 2004;15:205–213. PubMed

Wiley HS, Burke PM. Regulation of receptor tyrosine kinase signaling by endocytic trafficking. Traffic. 2001;2:12–18. PubMed

Mori S, Tanaka K, Omura S, Saito Y. Degradation process of ligand-stimulated platelet-derived growth factor beta-receptor involves ubiquitin-proteasome proteolytic pathway. J Biol Chem. 1995;270:29447–29452. PubMed

Chen KT, Lin JD, Liou MJ, Weng HF, Chang CA, Chan EC. An aberrant autocrine activation of the platelet-derived growth factor alpha-receptor in follicular and papillary thyroid carcinoma cell lines. Cancer Lett. 2006;231:192–205. PubMed

Wagner SA, Beli P, Weinert BT, Scholz C, Kelstrup CD, Young C, Nielsen ML, et al. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics. 2012;11:1578–1585. PubMed PMC

Baixauli F, Lopez-Otin C, Mittelbrunn M. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol. 2014;5:403. PubMed PMC

Piper RC, Katzmann DJ. Biogenesis and function of multivesicular bodies. Annu Rev Cell Dev Biol. 2007;23:519–547. PubMed PMC

Smith VL, Jackson L, Schorey JS. Ubiquitination as a Mechanism To Transport Soluble Mycobacterial and Eukaryotic Proteins to Exosomes. J Immunol. 2015;195:2722–2730. PubMed PMC

Feng H, Liu KW, Guo P, Zhang P, Cheng T, McNiven MA, Johnson GR, et al. Dynamin 2 mediates PDGFRalpha-SHP-2-promoted glioblastoma growth and invasion. Oncogene. 2012;31:2691–2702. PubMed PMC

Chen YN, LaMarche MJ, Chan HM, Fekkes P, Garcia-Fortanet J, Acker MG, Antonakos B, et al. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases. Nature. 2016;535:148–152. PubMed

Tajan M, de Rocca Serra A, Valet P, Edouard T, Yart A. SHP2 sails from physiology to pathology. Eur J Med Genet. 2015;58:509–525. PubMed

Higginbotham JN, Zhang Q, Jeppesen DK, Scott AM, Manning HC, Ochieng J, Franklin JL, et al. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting. J Extracell Vesicles. 2016;5:29254. PubMed PMC

S ELA, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12:347–357. PubMed

Edgar JR, Manna PT, Nishimura S, Banting G, Robinson MS. Tetherin is an exosomal tether. Elife. 2016;5 PubMed PMC

Villarroya-Beltri C, Baixauli F, Mittelbrunn M, Fernandez-Delgado I, Torralba D, Moreno-Gonzalo O, Baldanta S, et al. ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins. Nat Commun. 2016;7:13588. PubMed PMC

Pituch KC, Moyano AL, Lopez-Rosas A, Marottoli FM, Li G, Hu C, van Breemen R, et al. Dysfunction of platelet-derived growth factor receptor alpha (PDGFRalpha) represses the production of oligodendrocytes from arylsulfatase A-deficient multipotential neural precursor cells. J Biol Chem. 2015;290:7040–7053. PubMed PMC

Hyenne V, Apaydin A, Rodriguez D, Spiegelhalter C, Hoff-Yoessle S, Diem M, Tak S, et al. RAL-1 controls multivesicular body biogenesis and exosome secretion. J Cell Biol. 2015;211:27–37. PubMed PMC

McKenzie AJ, Hoshino D, Hong NH, Cha DJ, Franklin JL, Coffey RJ, Patton JG, et al. KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes. Cell Rep. 2016;15:978–987. PubMed PMC

Zuccato E, Blott EJ, Holt O, Sigismund S, Shaw M, Bossi G, Griffiths GM. Sorting of Fas ligand to secretory lysosomes is regulated by mono-ubiquitylation and phosphorylation. J Cell Sci. 2007;120:191–199. PubMed

Aceto N, Sausgruber N, Brinkhaus H, Gaidatzis D, Martiny-Baron G, Mazzarol G, Confalonieri S, et al. Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop. Nat Med. 2012;18:529–537. PubMed

Han T, Xiang DM, Sun W, Liu N, Sun HL, Wen W, Shen WF, et al. PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients. J Hepatol. 2015;63:651–660. PubMed

Baumdick M, Bruggemann Y, Schmick M, Xouri G, Sabet O, Davis L, Chin JW, et al. EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling. Elife. 2015;4 PubMed PMC

Song X, Ding Y, Liu G, Yang X, Zhao R, Zhang Y, Zhao X, et al. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases. J Biol Chem. 2016;291:8453–8464. PubMed PMC

Chen L, Brigstock DR. Integrins and heparan sulfate proteoglycans on hepatic stellate cells (HSC) are novel receptors for HSC-derived exosomes. FEBS Lett. 2016;590:4263–4274. PubMed PMC

Glycolysis in hepatic stellate cells coordinates fibrogenic extracellular vesicle release spatially to amplify liver fibrosis

Machine Learning-Based Classification of Abnormal Liver Tissues Using Relative Permittivity