基质金属蛋白酶-9调控CD73自视网膜色素上皮细胞膜表面脱落机制的初步研究

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

目的探讨基质金属蛋白酶（MMP）-9调控CD73自视网膜色素上皮（RPE）细胞膜表面脱落的机制。方法以脂多糖及肿瘤坏死因子-α共同干预体外培养的人RPE细胞，诱导RPE细胞膜表面CD73脱落。同时给予磷脂酶C（PLC）抑制剂ET-18-OCH，30．0gmol／L或广谱MMP抑制剂ONO-48175．0gmol／L进行干预；流式细胞仪检测RPE细胞膜表面CD73含量变化。以选择性MMP．2、MMP-9抑制剂或相应siRNA进行干预；利用基因定点突变技术改造CD73中潜在的MMP．9识别位点，在CD73^-/-。RPE细胞中过表达野生型（Wt）及突变型（Mut）CD73，检Nwt、Mut．CD73脱落情况。结果广谱MMP抑制剂可有效抑制CD73自RPE细胞膜表面脱落；PLC抑制剂不能抑制CD73自RPE细胞膜表面脱落。选择性抑制MMP-9活性或以siRNAT调MMP-9的表达也均可抑制CD73自RPE细胞膜表面脱落。表达于CD73。RPE细胞膜表面的Wt．CD73可发生脱落，而Lys^547Phe^548编码位点突变的CD73未见脱落。结论MMP．9通过水解Lys^547Phe^548位点而参与炎症状态下CD73自RPE细胞膜表面的脱落。

Objective To study how CD73 is shed from the retinal pigment epithelium （RPE） surface. Methods CD73 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. Results LPS 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 Lyss47- Phe548 sites mutant CD73. Conclusion MMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.