Matrix metalloproteinase-9 regulates the shedding of CD73 from retinal pigment epithelium_Chinese Journal of Ocular Fundus Diseases

Authors：

Kong Fanqiang , Zhou Shumin ,  Chen Song

Laboratory Medicine (Kong Fanqiang),Department of Ophtalmology (Chen Song), General Hospital of Tianjin Medical University, Tianjin 300052, China; Laboratory Medicine, The Second Hospital of Tianjin Medical University, Tianjin 300211, China (Zhou Shumin);

Corresponding author：

Chen Song, Email: chszyy248@163.com

Keywords：

Matrix metalloproteinase 9/antagonists & inhibitors; 5'-Nucleotidase; Retinal pigment epithelium

DOI：

10.3760/cma.j.issn.1005-1015.2017.05.018

Video：

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ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys⁵⁴⁷-Phe⁵⁴⁸ coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73^-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73^-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys⁵⁴⁷-Phe⁵⁴⁸ sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys⁵⁴⁷ -Phe⁵⁴⁸ sites.

Citation： Kong Fanqiang, Zhou Shumin, Chen Song. Matrix metalloproteinase-9 regulates the shedding of CD73 from retinal pigment epithelium. Chinese Journal of Ocular Fundus Diseases, 2017, 33(5): 518-522. doi: 10.3760/cma.j.issn.1005-1015.2017.05.018 Copy

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1. Elliott MR, Chekeni FB, Trampont PC, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[J]. Nature,2009,461(7261):282-286.DOI: 10.1038/nature08296.
2. Stagg J, Smyth MJ. Extracellular adenosine triphosphate and adenosine in cancer[J]. Oncogene, 2010,29(39):5346-5358.DOI:10.1038/onc.2010.292.
3. Velasquez S, Malik S, Lutz SE, et al. Pannexin1 Channels are required for chemokine-mediated migration of CD4+ T lymphocytes: role in inflammation and experimental autoimmune encephalomyelitis[J]. J Immunol, 2016,196(10):4338-4347. DOI: 10.4049/jimmunol.1502440.
4. Pandolfi JB, Ferraro AA, Sananez I, et al. ATP-induced inflammation drives tissue-resident Th17 cells in metabolically unhealthy obesity[J]. J Immunol, 2016,196(8):3287-3296. DOI: 10.4049/jimmunol.1502506.
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7. Li N, Mu L, Wang J, et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune myasthenia gravis severity[J]. Eur J Immunol, 2012,42(5):1140-1151. DOI: 10.1002/eji.201142088.
8. Yao SQ, Li ZZ, Huang QY, et al. Genetic inactivation of the adenosine A(2A) receptor exacerbates brain damage in mice with experimental autoimmune encephalomyelitis[J]. J Neurochem,2012,123(1):100-112. DOI: 10.1111/j.1471-4159.2012.07807.x.
9. Fletcher JM, Lonergan R, Costelloe L, et al. CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis[J]. J Immunol,2009,183(11):7602-7610.
10. Saze Z, Schuler PJ, Hong CS, et al. Adenosine production by human B cells and B cell-mediated suppression of activated T cells[J]. Blood, 2013,122(1):9-18.DOI: 10.1182/blood-2013-02-482406.
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14. Yamamoto A, Yano S, Shiraga M, et al. A third-generation matrix metalloproteinase (MMP) inhibitor (ONO-4817) combined with docetaxel suppresses progression of lung micrometastasis of MMP-expressing tumor cells in nude mice. Int J Cancer, 2003, 103(6):822-828. DOI: 10.1002/ijc.10875.
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21. Hollborn M, Stathopoulos C, Steffen A, et al. Positive feedback regulation between MMP-9 and VEGF in human RPE cells[J]. Invest Ophthalmol Vis Sci, 2007,48(9):4360-4367. DOI: 10.1167/iovs.06-1234.
22. Hou X, Han QH, Hu D, et al. Mechanical force enhances MMP-2 activation via p38 signaling pathway in human retinal pigment epithelial cells[J]. Graefe’s Arch Clin Exp Ophthalmol, 2009,247(11):1477-1486. DOI: 10.1007/s00417-009-1135-1.

Chinese Journal of Ocular Fundus Diseases

Matrix metalloproteinase-9 regulates the shedding of CD73 from retinal pigment epithelium

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