Purinergic Signalling

, Volume 14, Issue 4, pp 443–457 | Cite as

Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation

  • Wei Zhang
  • Shumin Zhou
  • Guoping Liu
  • Fanqiang Kong
  • Song ChenEmail author
  • Hua Yan
Original Article


Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE’s immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.


CD73 Shedding Lipid rafts MMP-9 Adenosine receptor A1 



This study was supported by the National Natural Science Foundation of China (No. 81570833).

Compliance with ethical standards

Institutional approval was obtained, and institutional guidelines regarding animal experimentation were followed.

Conflicts of interest

Wei Zhang declares that he has no conflict of interest.

Shumin Zhou declares that she has no conflict of interest.

Guoping Liu declares that he has no conflict of interest.

Fanqiang Kong declares that he has no conflict of interest.

Song Chen declares that he has no conflict of interest.

Supplementary material

11302_2018_9628_MOESM1_ESM.docx (77 kb)
ESM 1 (DOCX 77 kb)


  1. 1.
    Yegutkin GG (2008) Nucleotide- and nucleoside-converting ectoenzymes: important modulators of purinergic signalling cascade. Biochim Biophys Acta 1783:673–694CrossRefGoogle Scholar
  2. 2.
    Idzko M, Ferrari D, Eltzschig HK (2014) Nucleotide signalling during inflammation. Nature 509:310–317CrossRefGoogle Scholar
  3. 3.
    Zarek PE, Huang C, Lutz ER, Kowalski J, Horton MR, Linden J (2008) A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells. Blood 111:251–259CrossRefGoogle Scholar
  4. 4.
    Junger WG (2011) Immune cell regulation by autocrine purinergic signalling. Nat Rev Immunol 11:201–212CrossRefGoogle Scholar
  5. 5.
    Smyth L.A, Ratnasothy K, Tsang JY, Boardman D, Warley A, Lechler R, Lombardi G (2013) CD73 expression on extracellular vesicles derived from CD4+ CD25+ Foxp3+ T cells contributes to their regulatory function. Eur J Immunol 43(9):2430–2440CrossRefGoogle Scholar
  6. 6.
    Takedachi M, Qu D, Ebisuno Y, Oohara H, Joachims ML, McGee ST, Maeda E, McEver RP, Tanaka T, Miyasaka M, Murakami S, Krahn T, Blackburn MR, Thompson LF (2008) CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes. J Immunol 180(9):6288–6296CrossRefGoogle Scholar
  7. 7.
    Coffey F, Lee SY, Buus TB, Lauritsen JP, Wong GW, Joachims ML, Thompson LF, Zúñiga-Pflücker JC, Kappes DJ, Wiest DL (2014) The TCR ligand-inducible expression of CD73 marks γδ lineage commitment and a metastable intermediate in effector specification. J Exp Med 211(2):329–343CrossRefGoogle Scholar
  8. 8.
    Chen S, Zhou S, Zang K, Kong F, Liang D, Yan H (2014) CD73 expression in RPE cells is associated with the suppression of conventional CD4 cell proliferation. Exp Eye Res 127:26–36CrossRefGoogle Scholar
  9. 9.
    Dong K, Gao ZW, Zhang HZ (2016) The role of adenosinergic pathway in human autoimmune diseases. Immunol Res 64:1133–1141CrossRefGoogle Scholar
  10. 10.
    Regateiro FS, Cobbold SP, Waldmann H (2013) CD73 and adenosine generation in the creation of regulatory microenvironments. Clin Exp Immunol 171:1–7CrossRefGoogle Scholar
  11. 11.
    Chrobak P, Charlebois R, Rejtar P, El Bikai R, Allard B, Stagg J (2015) CD73 plays a protective role in collagen-induced arthritis. J Immunol 194:2487–2492CrossRefGoogle Scholar
  12. 12.
    Botta Gordon-Smith S, Ursu S, Eaton S, Moncrieffe H, Wedderburn LR (2015) Correlation of low CD73 expression on synovial lymphocytes with reduced adenosine generation and higher disease severity in juvenile idiopathic arthritis. Arthritis Rheumatol 67:545–554CrossRefGoogle Scholar
  13. 13.
    Li J, Wang L, Chen X, Li L, Li Y, Ping Y, Huang L, Yue D, Zhang Z, Wang F, Li F, Yang L, Huang J, Yang S, Li H, Zhao X, Dong W, Yan Y, Zhao S, Huang B, Zhang B, Zhang Y (2017) CD39/CD73 upregulation on myeloid-derived suppressor cells via TGF-β-mTOR-HIF-1 signaling in patients with non-small cell lung cancer. Oncoimmunology 6:e1320011CrossRefGoogle Scholar
  14. 14.
    Whiteside TL (2017) Targeting adenosine in cancer immunotherapy: a review of recent progress. Expert Rev Anticancer Ther 17:527–535CrossRefGoogle Scholar
  15. 15.
    Allard B, Longhi MS, Robson SC, Stagg J (2017) The ectonucleotidases CD39 and CD73: novel checkpoint inhibitor targets. Immunol Rev 276:121–144CrossRefGoogle Scholar
  16. 16.
    Young A, Ngiow SF, Barkauskas DS, Sult E, Hay C, Blake SJ, Huang Q, Liu J, Takeda K, Teng MW, Sachsenmeier K, Smyth MJ (2016) Co-inhibition of CD73 and A2AR adenosine signaling improves anti-tumor immune responses. Cancer Cell 30:391–403CrossRefGoogle Scholar
  17. 17.
    Doyle JW, Dowgiert RK, Buzney SM (1995) Retinoic acid metabolism in cultured retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 36:708–717PubMedGoogle Scholar
  18. 18.
    Chen M, Muckersie E, Robertson M, Fraczek M, Forrester JV, Xu H (2008) Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07. Invest Ophthalmol Vis Sci 49:3699–3706CrossRefGoogle Scholar
  19. 19.
    Santerre RF, Allen NE, Hobbs JN Jr, Rao RN, Schmidt RJ (1984) Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells. Gene 30:147–156CrossRefGoogle Scholar
  20. 20.
    Macdonald JL, Pike LJ (2005) A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 46:1061–1067CrossRefGoogle Scholar
  21. 21.
    Aplin FP, Luu CD, Vessey KA, Guymer RH, Shepherd RK, Fletcher EL (2014) ATP-induced photoreceptor death in a feline model of retinal degeneration. Invest Ophthalmol Vis Sci 55(12):8319–8329CrossRefGoogle Scholar
  22. 22.
    Lu W, Hu H, Sévigny J, Gabelt BT, Kaufman PL, Johnson EC, Morrison JC, Zode GS, Sheffield VC, Zhang X, Laties AM, Mitchell CH (2015) Rat, mouse, and primate models of chronic glaucoma show sustained elevation of extracellular ATP and altered purinergic signaling in the posterior eye. Invest Ophthalmol Vis Sci 56(5):3075–3083CrossRefGoogle Scholar
  23. 23.
    Sakamoto K, Endo K, Suzuki T, Fujimura K, Kurauchi Y, Mori A, Nakahara T, Ishii K (2015) P2X7 receptor antagonists protect against N-methyl-D-aspartic acid-induced neuronal injury in the rat retina. Eur J Pharmacol 756:52–58CrossRefGoogle Scholar
  24. 24.
    Bar-Yehuda S, Luger D, Ochaion A, Cohen S, Patokaa R, Zozulya G, Silver PB, de Morales JM, Caspi RR, Fishman P (2011) Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101. Int J Mol Med 28(5):727–731PubMedPubMedCentralGoogle Scholar
  25. 25.
    Avni I, Garzozi HJ, Barequet IS, Segev F, Varssano D, Sartani G, Chetrit N, Bakshi E, Zadok D, Tomkins O, Litvin G, Jacobson KA, Fishman S, Harpaz Z, Farbstein M, Yehuda SB, Silverman MH, Kerns WD, Bristol DR, Cohn I, Fishman P (2010) Treatment of dry eye syndrome with orally administered CF101: data from a phase 2 clinical trial. Ophthalmology 117(7):1287–1293CrossRefGoogle Scholar
  26. 26.
    Jacobson KA, Civan MM (2016) Ocular purine receptors as drug targets in the eye. J Ocul Pharmacol Ther 32(8):534–547CrossRefGoogle Scholar
  27. 27.
    Arab S, Kheshtchin N, Ajami M, Ashurpoor M, Safvati A, Namdar A, Mirzaei R, Mousavi Niri N, Jadidi-Niaragh F, Ghahremani MH, Hadjati J (2017) Increased efficacy of a dendritic cell-based therapeutic cancer vaccine with adenosine receptor antagonist and CD73 inhibitor. Tumour Biol 39(3):1010428317695021CrossRefGoogle Scholar
  28. 28.
    Allard B, Turcotte M, Stagg J (2014) 2014.Targeting CD73 and downstream adenosine receptor signaling in triple-negative breast cancer. Expert Opin Ther Targets 18:863–881CrossRefGoogle Scholar
  29. 29.
    Beavis PA, Stagg J, Darcy PK, Smyth MJ (2012) CD73: a potent suppressor of antitumor immune responses. Trends Immunol 33(5):231–237CrossRefGoogle Scholar
  30. 30.
    Stagg J, Beavis PA, Divisekera U, Liu MC, Möller A, Darcy PK, Smyth MJ (2012) CD73-deficient mice are resistant to carcinogenesis. Cancer Res 72(9):2190–2196CrossRefGoogle Scholar
  31. 31.
    Fausther M, Lavoie EG, Goree JR, Dranoff JA (2017) An Elf2-like transcription factor acts as repressor of the mouse ecto-5′-nucleotidase gene expression in hepatic myofibroblasts. Purinergic Signal 13(4):417–428CrossRefGoogle Scholar
  32. 32.
    Kao DJ, Saeedi BJ, Kitzenberg D, Burney KM, Dobrinskikh E, Battista KD, Vázquez-Torres A, Colgan SP, Kominsky DJ (2017) Intestinal epithelial Ecto-5'-Nucleotidase (CD73) regulates intestinal colonization and infection by nontyphoidal salmonella. Infect Immun 85(10)Google Scholar
  33. 33.
    Kling L, Benck U, Breedijk A, Leikeim L, Heitzmann M, Porubsky S, Krämer BK, Yard BA, Kälsch AI (2017) Changes in CD73, CD39 and CD26 expression on T-lymphocytes of ANCA-associated vasculitis patients suggest impairment in adenosine generation and turn-over. Sci Rep 7(1):11683CrossRefGoogle Scholar
  34. 34.
    Monguió-Tortajada M, Roura S, Gálvez-Montón C, Franquesa M, Bayes-Genis A, Borràs FE (2017) Mesenchymal stem cells induce expression of CD73 in human monocytes in vitro and in a swine model of myocardial infarction in vivo. Front Immunol 8:1577CrossRefGoogle Scholar
  35. 35.
    Airas L, Niemelä J, Salmi M, Puurunen T, Smith DJ, Jalkanen S (1997) Differential regulation and function of CD73, a glycosyl-phosphatidylinositol-linked 70-kD adhesion molecule, on lymphocytes and endothelial cells. J Cell Biol 136(2):421–431CrossRefGoogle Scholar
  36. 36.
    Kothekar D, Bandivdekar A, Dasgupta D (2014) Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C. Indian J Biochem Biophys 51(4):263–270PubMedGoogle Scholar
  37. 37.
    Yadav M, Raghupathy R, Saikam V, Dara S, Singh PP, Sawant SD, Mayor S, Vishwakarma RA (2014) Synthesis of non-hydrolysable mimics of glycosylphosphatidylinositol (GPI) anchors. Org Biomol Chem 12(7):1163–1172CrossRefGoogle Scholar
  38. 38.
    Maksimow M, Kyhälä L, Nieminen A, Kylänpää L, Aalto K, Elima K, Mentula P, Lehti M, Puolakkainen P, Yegutkin GG, Jalkanen S, Repo H, Salmi M (2014) Early prediction of persistent organ failure by soluble CD73 in patients with acute pancreatitis*. Crit Care Med 42(12):2556–2564CrossRefGoogle Scholar
  39. 39.
    Morello S, Capone M, Sorrentino C, Giannarelli D, Madonna G, Mallardo D, Grimaldi AM, Pinto A, Ascierto PA (2017) Soluble CD73 as biomarker in patients with metastatic melanoma patients treated with nivolumab. J Transl Med 15(1):244CrossRefGoogle Scholar
  40. 40.
    Shen Q, Bourdais G, Pan H, Robatzek S, Tang D (2017) Arabidopsis glycosylphosphatidylinositol-anchored protein LLG1 associates with and modulates FLS2 to regulate innate immunity. Proc Natl Acad Sci U S A 1142:5749–5754CrossRefGoogle Scholar
  41. 41.
    Tiengwe C, Bush PJ, Bangs JD (2017) Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes. PLoS Pathog 13(5):e1006366CrossRefGoogle Scholar
  42. 42.
    Augusto E, Matos M, Sévigny J, El-Tayeb A, Bynoe MS, Müller CE, Cunha RA, Chen JF (2013) Ecto-5′-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions. J Neurosci 33(28):11390–11399CrossRefGoogle Scholar
  43. 43.
    Hart ML, Grenz A, Gorzolla IC, Schittenhelm J, Dalton JH, Eltzschig HK (2011) Hypoxia-inducible factor-1α-dependent protection from intestinal ischemia/reperfusion injury involves ecto-5′-nucleotidase (CD73) and the A2B adenosine receptor. J Immunol 186(7):4367–4374CrossRefGoogle Scholar
  44. 44.
    Helms JB, Zurzolo C (2004) Lipids as targeting signals: lipid rafts and intracelular trafficking. Traffic 5:247–254CrossRefGoogle Scholar
  45. 45.
    Cinek T, Horejsi V (1992) The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol 149:2262–2270PubMedGoogle Scholar
  46. 46.
    Sargiacomo M, Sudol M, Tang Z, Lisanti MP (1993) Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol 122:789–807CrossRefGoogle Scholar
  47. 47.
    Zurzolo C, van't Hof W, van Meer G, Rodriguez-Boulan E (1994) VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells. EMBO J 13:42–53CrossRefGoogle Scholar
  48. 48.
    Wolczyk D, Zaremba-Czogalla M, Hryniewicz-Jankowska A, Tabola R, Grabowski K, Sikorski AF, Augoff K (2016) TNF-α promotes breast cancer cell migration and enhances the concentration of membrane-associated proteases in lipid rafts. Cell Oncol (Dordr) 39(4):353–363CrossRefGoogle Scholar
  49. 49.
    Tewari R, Choudhury SR, Mehta VS, Sen E (2012) TNFα regulates the localization of CD40 in lipid rafts of glioma cells. Mol Biol Rep 39(9):8695–8699Google Scholar
  50. 50.
    Schoeniger A, Fuhrmann H, Schumann J (2016) LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition. PeerJ 4:e1663CrossRefGoogle Scholar
  51. 51.
    Rittiner JE, Korboukh I, Hull-Ryde EA, Jin J, Janzen WP, Frye SV, Zylka MJ (2012) AMP is an adenosine A1 receptor agonist. J Biol Chem 287(8):5301–5309CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Wei Zhang
    • 1
  • Shumin Zhou
    • 2
  • Guoping Liu
    • 3
  • Fanqiang Kong
    • 4
  • Song Chen
    • 4
    Email author
  • Hua Yan
    • 4
  1. 1.Department of StrabismusTianjin Eye Disease HospitalTianjinChina
  2. 2.Clinical laboratoryThe 2nd hospital of Tianjin Medical UniversityTianjinChina
  3. 3.Department of NeurologyTianjin first central hospitalTianjinChina
  4. 4.General hospital of Tianjin Medical UniversityTianjinChina

Personalised recommendations