Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Fundus Diseases, Shanghai, China.
Curr Mol Med. 2019;19(6):395-404. doi: 10.2174/1566524019666190509105502.
Bcl-2/adenovirus E1B-19kDa-interacting protein (BNIP3), an important target of hypoxia-inducible factors-1 alpha (HIF-1α), was reported to be overexpressed under hypoxic condition. Our previous study demonstrated the protective effect on detached retina by BNIP3-mediated autophagy. The study investigated the role of BNIP3-mediated autophagy in retinal pigment epithelial (RPE) cells under hypoxia, and observed the relationship between BNIP3, vascular endothelial growth factor (VEGF) and inflammatory response in hypoxic RPE cells.
BNIP3 knock down in retinal pigment epithelial cells was performed by small interfering RNA (siRNA) technology in ARPE-19 cells, a human RPE cell line. Both control and BNIP3-knockdown ARPE-19 cells were then subjected to a hypoxic challenge using cobalt (II) chloride (CoCl2). The expression of autophagy-related genes, VEGF and inflammatory factors (IL-18, IL-8, MMP-2, MMP-9, NLRP3, TNF-α) in RPE cells was examined using quantitative Polymerase Chain Reaction (qPCR). The protein levels of HIF-1α, BNIP3, the maker proteins (ATG5, LC3,p62, Beclin-1) of autophagy and the component proteins (p-p70S6K, p70S6K, mTOR, p-mTOR) of the mTORC1 pathway were analyzed by Western blot. BNIP3 subcellualr localization was detected by immunofluorescence. Cell viability was measured with Cell Counting kit-8. Cell apoptosis was examined by TUNEL staining and caspase-3 activity assay.
The expression levels of BNIP3, HIF-1α and marker genes of autophagy were upregulated in ARPE-19 cells in response to hypoxia. Importantly, hypoxia-induced autophagy was mediated by the mTORC1 pathway, and was blocked upon BNIP3 knockdown. Additionally, hypoxia reduced cell viability, which was relieved by an mTORC1 inhibitor. Also, autophagy protected ARPE-19 cells from CoCl2-induced cell apoptosis. Moreover, inhibition of autophagy upregulated the expression of VEGF and IL-18, and downregulated the expression of other inflammatory factors in the hypoxic ARPE-19 cells.
BNIP3-mediated autophagy under hypoxia is involved in regulating inflammatory response and VEGF expression, which consequently affects the cell viability of RPE cells.
Bcl-2/腺病毒 E1B-19kDa 相互作用蛋白（BNIP3）是缺氧诱导因子-1α（HIF-1α）的重要靶标，据报道在缺氧条件下过度表达。我们之前的研究表明 BNIP3 介导的自噬对分离视网膜具有保护作用。本研究探讨了 BNIP3 介导的自噬在缺氧状态下对视网膜色素上皮（RPE）细胞的作用，并观察了缺氧 RPE 细胞中 BNIP3、血管内皮生长因子（VEGF）和炎症反应之间的关系。
采用小干扰 RNA（siRNA）技术在人 RPE 细胞系 ARPE-19 细胞中敲低 BNIP3。然后用氯化钴（CoCl2）对对照和 BNIP3 敲低的 ARPE-19 细胞进行缺氧处理。采用实时定量聚合酶链反应（qPCR）检测 RPE 细胞中自噬相关基因、VEGF 和炎症因子（IL-18、IL-8、MMP-2、MMP-9、NLRP3、TNF-α）的表达。采用 Western blot 分析 HIF-1α、BNIP3、自噬标志物蛋白（ATG5、LC3、p62、Beclin-1）和 mTORC1 通路组成蛋白（p-p70S6K、p70S6K、mTOR、p-mTOR）的蛋白水平。通过免疫荧光检测 BNIP3 亚细胞定位。采用细胞计数试剂盒-8 检测细胞活力。通过 TUNEL 染色和 caspase-3 活性测定检测细胞凋亡。
缺氧诱导 ARPE-19 细胞中 BNIP3、HIF-1α 和自噬标志物基因的表达上调。重要的是，缺氧诱导的自噬是通过 mTORC1 通路介导的，而 BNIP3 敲低则阻断了自噬。此外，缺氧降低了细胞活力，而 mTORC1 抑制剂可缓解这种作用。此外，自噬可保护 ARPE-19 细胞免受 CoCl2 诱导的细胞凋亡。此外，自噬抑制上调了缺氧 ARPE-19 细胞中 VEGF 和 IL-18 的表达，下调了其他炎症因子的表达。
缺氧下 BNIP3 介导的自噬参与调节炎症反应和 VEGF 表达，从而影响 RPE 细胞的细胞活力。
