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

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Abstract

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.

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Acknowledgments

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

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Authors and Affiliations

  1. Department of Strabismus, Tianjin Eye Disease Hospital, Tianjin, 300020, China

    Wei Zhang

  2. Clinical laboratory, The 2nd hospital of Tianjin Medical University, Tianjin, 300211, China

    Shumin Zhou

  3. Department of Neurology, Tianjin first central hospital, Tianjin, 300192, China

    Guoping Liu

  4. General hospital of Tianjin Medical University, Tianjin, 300052, China

    Fanqiang Kong, Song Chen & Hua Yan

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  1. Wei Zhang
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  2. Shumin Zhou
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Correspondence to Song Chen.

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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.

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Zhang, W., Zhou, S., Liu, G. et al. Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation. Purinergic Signalling 14, 443–457 (2018). https://doi.org/10.1007/s11302-018-9628-1

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