Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation
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.
KeywordsCD73 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.
- 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
- 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
- 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
- 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
- 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
- 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