BNIP3-mediated Autophagy Induced Inflammatory Response and Inhibited VEGF Expression in Cultured Retinal Pigment Epithelium Cells Under Hypoxia.
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.
['Chen|Yuhong|Y|', 'Yan|Quan|Q|', 'Xu|Yihua|Y|', 'Ye|Fuxiang|F|', 'Sun|Xiaodong|X|', 'Zhu|Hong|H|', 'Wang|Hong|H|']
D001343:Autophagy D015415:Biomarkers D015687:Cell Hypoxia D002460:Cell Line D002470:Cell Survival D002478:Cells, Cultured D003035:Cobalt D005786:Gene Expression Regulation D006801:Humans D051795:Hypoxia-Inducible Factor 1, alpha Subunit D008565:Membrane Proteins D011518:Proto-Oncogene Proteins D034622:RNA Interference D055213:Retinal Pigment Epithelium D015398:Signal Transduction D042461:Vascular Endothelial Growth Factor A
autophagy VEGF hypoxia BNIP3 RPEs inflammation.