BNIP3-mediated Autophagy Induced Inflammatory Response and Inhibited VEGF Expression in Cultured Retinal Pigment Epithelium Cells Under Hypoxia

Author(s): Yuhong Chen*, Quan Yan*, Yihua Xu, Fuxiang Ye, Xiaodong Sun, Hong Zhu, Hong Wang.

Journal Name: Current Molecular Medicine

Volume 19 , Issue 6 , 2019

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Abstract:

Background: 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.

Methods: 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.

Results: 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.

Conclusion: BNIP3-mediated autophagy under hypoxia is involved in regulating inflammatory response and VEGF expression, which consequently affects the cell viability of RPE cells.

Keywords: BNIP3, hypoxia, RPEs, autophagy, VEGF, inflammation.

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Article Details

VOLUME: 19
ISSUE: 6
Year: 2019
Page: [395 - 404]
Pages: 10
DOI: 10.2174/1566524019666190509105502
Price: $58

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