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Current Molecular Medicine


ISSN (Print): 1566-5240
ISSN (Online): 1875-5666

Research Article

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 and Hong Wang

Volume 19, Issue 6, 2019

Page: [395 - 404] Pages: 10

DOI: 10.2174/1566524019666190509105502

Price: $65


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.

Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med 2008; 358(24): 2606-17.
van Leeuwen R, Klaver CC, Vingerling JR, Hofman A, de Jong PT. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol 2003; 18(9): 845-54.
Hernandez-Zimbron LF, Zamora-Alvarado R, Ochoa-De la Paz L, et al. Age-related macular degeneration: New paradigms for treatment and management of AMD. Oxid Med Cell Longev 2018; 2018: 8374647.
Boulton M, Dayhaw-Barker P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye (London, England) 2001; 15(Pt 3): 384-9.
Strauss O. The retinal pigment epithelium in visual function. Physiol Rev 2005; 85(3): 845-81.
Whitmore SS, Sohn EH, Chirco KR, et al. Complement activation and choriocapillaris loss in early AMD: implications for pathophysiology and therapy. Prog Retin Eye Res 2015; 45: 1-29.
Risk factors associated with age-related macular degeneration. A case-control study in the age-related eye disease study: Age-Related Eye Disease Study Report Number 3. Ophthalmology 2000; 107(12): 2224-32.
Marneros AG, Fan J, Yokoyama Y, et al. Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function. Am J Pathol 2005; 167(5): 1451-9.
Schlingemann RO. Role of growth factors and the wound healing response in age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol 2004; 242(1): 91-101.
Zhao W, Wang YS, Hui YN, et al. Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1alpha with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system. Graefes Arch Clin Exp Ophthalmol 2008; 246(10): 1413-22.
Hardy P, Beauchamp M, Sennlaub F, et al. New insights into the retinal circulation: inflammatory lipid mediators in ischemic retinopathy. Prostaglandins Leukot Essent Fatty Acids 2005; 72(5): 301-25.
Doherty J, Baehrecke EH. Life, death and autophagy. Nat Cell Biol 2018; 20(10): 1110-7.
Samie M, Lim J, Verschueren E, et al. Selective autophagy of the adaptor TRIF regulates innate inflammatory signaling. Nat Immunol 2018; 19(3): 246-54.
Zou J, Chen Z, Wei X, et al. Cystatin C as a potential therapeutic mediator against Parkinson’s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units. Cell Death Dis 2017; 8(6): e2854.
Janji B, Berchem G, Chouaib S. Targeting Autophagy in the Tumor Microenvironment: New Challenges and Opportunities for Regulating Tumor Immunity. Front Immunol 2018; 9: 887.
Lobo MR, Green SC, Schabel MC, Gillespie GY, Woltjer RL, Pike MM. Quinacrine synergistically enhances the antivascular and antitumor efficacy of cediranib in intracranial mouse glioma. Neuro-oncol 2013; 15(12): 1673-83.
Kim JH, Kim JH, Yu YS, Mun JY, Kim KW. Autophagy-induced regression of hyaloid vessels in early ocular development. Autophagy 2010; 6(7): 922-8.
Zhang P, Wang Y, Hui Y, et al. Inhibition of VEGF expression by targeting HIF-1 alpha with small interference RNA in human RPE cells. Ophthalmologica 2007; 221(6): 411-7.
Forooghian F, Razavi R, Timms L. Hypoxia-inducible factor expression in human RPE cells. Br J Ophthalmol 2007; 91(10): 1406-10.
Cho KS, Yoon YH, Choi JA, Lee SJ, Koh JY. Induction of autophagy and cell death by tamoxifen in cultured retinal pigment epithelial and photoreceptor cells. Invest Ophthalmol Vis Sci 2012; 53(9): 5344-53.
Blasiak J, Petrovski G, Vereb Z, Facsko A, Kaarniranta K. Oxidative stress, hypoxia, and autophagy in the neovascular processes of age-related macular degeneration. BioMed Res Int 2014; 2014: 768026.
Li Y, Shen H, Shi J, Tang L. The effects of alpha lipoic acid in preventing oxidative stress-induced retinal pigment epithelial cell injury. Can J Physiol Pharmacol 2014; 92(9): 765-72.
Liu H, Zhu H, Li T, Zhang P, Wang N, Sun X. Prolyl-4-Hydroxylases Inhibitor Stabilizes HIF-1alpha and Increases Mitophagy to Reduce Cell Death After Experimental Retinal Detachment. Invest Ophthalmol Vis Sci 2016; 57(4): 1807-15.
Gallo S, Gatti S, Sala V, et al. Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy. Cell Death Dis 2014; 5: e1185.
Zhang X, Chen L. Effects of CoCl2-simulated hypoxia on the expression levels of matrix metalloproteinases in renal adenocarcinoma cells and renal tubular epithelial cells. Exp Ther Med 2018; 16(2): 1454-60.
Liu GH, Wen Y, Yang P, Liu GF. Regulation by Pink1 on the mitochondrial dysfunction in endothelial cells post the hypoxia mimetic agent CoCl2 treatment. Eur Rev Med Pharmacol Sci 2018; 22(17): 5704-11.
Nunes SC, Lopes-Coelho F, Gouveia-Fernandes S, Ramos C, Pereira SA, Serpa J. Cysteine boosters the evolutionary adaptation to CoCl2 mimicked hypoxia conditions, favouring carboplatin resistance in ovarian cancer. BMC Evol Biol 2018; 18(1): 97.
Ma T, Patel H, Babapoor-Farrokhran S, et al. KSHV induces aerobic glycolysis and angiogenesis through HIF-1-dependent upregulation of pyruvate kinase 2 in Kaposi’s sarcoma. Angiogenesis 2015; 18(4): 477-88.
Yang FQ, Liu M, Yang FP, et al. VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1alpha. Mol Biol Rep 2014; 41(3): 1511-8.
De Francesco EM, Lappano R, Santolla MF, Marsico S, Caruso A, Maggiolini M. HIF-1alpha/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs). BCR 2013; 15(4): R64.
Zhang Y, Liu Q, Wang F, et al. Melatonin antagonizes hypoxia-mediated glioblastoma cell migration and invasion via inhibition of HIF-1alpha. J Pineal Res 2013; 55(2): 121-30.
Ney PA. Mitochondrial autophagy: Origins, significance, and role of BNIP3 and NIX. Biochim Biophys Acta 2015; 1853(10 Pt B): 2775-83.
Shi RY, Zhu SH, Li V, Gibson SB, Xu XS, Kong JM. BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke. CNS Neurosci Ther 2014; 20(12): 1045-55.
Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med 2013; 368(19): 1845-6.
Kaliszczak M, van Hechanova E, Li Y, et al. The HDAC6 inhibitor C1A modulates autophagy substrates in diverse cancer cells and induces cell death. Br J Cancer 2018; 119(10): 1278-87.
Assali A, Akhavan O, Mottaghitalab F, et al. Cationic graphene oxide nanoplatform mediates miR-101 delivery to promote apoptosis by regulating autophagy and stress. Int J Nanomedicine 2018; 13: 5865-86.
Arjamaa O, Nikinmaa M, Salminen A, Kaarniranta K. Regulatory role of HIF-1alpha in the pathogenesis of age-related macular degeneration (AMD). Ageing Res Rev 2009; 8(4): 349-58.
Stefansson E, Geirsdottir A, Sigurdsson H. Metabolic physiology in age related macular degeneration. Prog Retin Eye Res 2011; 30(1): 72-80.
Kent DL. Age-related macular degeneration: beyond anti-angiogenesis. Mol Vis 2014; 20: 46-55.
Bernard O, Jeny F, Uzunhan Y, et al. Mesenchymal stem cells reduce hypoxia-induced apoptosis in alveolar epithelial cells by modulating HIF and ROS hypoxic signaling. Am J Physiol Lung Cell Mol Physiol 2018; 314(3): L360-l71.
Radreau P, Rhodes JD, Mithen RF, Kroon PA, Sanderson J. Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells. Exp Eye Res 2009; 89(6): 995-1002.
Fordjour PA, Wang L, Gao H, et al. Targeting BNIP3 in inflammation-mediated heart failure: a novel concept in heart failure therapy. Heart Fail Rev 2016; 21(5): 489-97.
Li H, Miao W, Ma J, et al. Acute Exercise-Induced Mitochondrial Stress Triggers an Inflammatory Response in the Myocardium via NLRP3 Inflammasome Activation with Mitophagy. Oxid Med Cell Longev 2016; 2016: 1987149.
Wu SY, Lan SH, Cheng DE, et al. Ras-related tumorigenesis is suppressed by BNIP3-mediated autophagy through inhibition of cell proliferation. Neoplasia (New York, NY) 2011; 13(12): 1171-82.
Azad MB, Gibson SB. Role of BNIP3 in proliferation and hypoxia-induced autophagy: implications for personalized cancer therapies. Ann N Y Acad Sci 2010; 1210: 8-16.
Han B, Li W, Sun Y, Zhou L, Xu Y, Zhao X. A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces cell autophagy and apoptosis in esophageal squamous cell carcinoma cells via up-regulation of BNIP3 and N-myc downstream-regulated gene-1. PLoS One 2014; 9(9): e107204.
Kim HR, Luo Y, Li G, Kessel D. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection. Cancer Res 1999; 59(14): 3429-32.
Shimizu S, Tsujimoto Y. Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity. Proc Natl Acad Sci USA 2000; 97(2): 577-82.
Kelekar A, Thompson CB. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol 1998; 8(8): 324-30.
Mahalingaiah PK, Singh KP. Chronic oxidative stress increases growth and tumorigenic potential of MCF-7 breast cancer cells. PLoS One 2014; 9(1): e87371.
Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl J Med 2011; 365(6): 537-47.
Puissant A, Fenouille N, Auberger P. When autophagy meets cancer through p62/SQSTM1. Am J Cancer Res 2012; 2(4): 397-413.
Bensaad K, Cheung EC, Vousden KH. Modulation of intracellular ROS levels by TIGAR controls autophagy. The EMBO journal 2009; 28(19): 3015-26.
Scherz-Shouval R, Elazar Z. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011; 36(1): 30-8.
Bellot G, Garcia-Medina R, Gounon P, et al. Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol 2009; 29(10): 2570-81.
Maiuri MC, Le Toumelin G, Criollo A, et al. Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. The EMBO J 2007; 26(10): 2527-39.
Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122(6): 927-39.
Li Y, Wang Y, Kim E, et al. Bnip3 mediates the hypoxia-induced inhibition on mammalian target of rapamycin by interacting with Rheb. J Biol Chem 2007; 282(49): 35803-13.

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