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Charles B Wright, Bradley D Gelfand, Jayakrishna Ambati; Hypoxia signaling pathways in retinal pigment epithelium require Alu RNA. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5813.
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© ARVO (1962-2015); The Authors (2016-present)
Hypoxia is implicated in numerous visual diseases, including retinopathy of prematurity (ROP), diabetic retinopathy (DR), and age-related macular degeneration (AMD). It was previously found that retinal pigment epithelium (RPE) cell death in AMD is mediated by Alu RNA-induced NLRP3 inflammasome activation caused by reduced DICER1. Other reports also demonstrate hypoxia induces NLRP3 inflammasome activation and DICER1 suppression. We therefore sought to investigate whether hypoxia-induced NLRP3 inflammasome activation was Alu RNA-dependent.
Primary human retinal pigment epithelium (RPE) cells were collected as previously described. Hypoxic conditions were induced by housing cells in a low (1% and 5%) oxygen environment or by exposing cells to media containing CoCl2. Alu RNA accumulation was assayed by northern blot and protein expression assayed by western blot. Alu RNA blockade was achieved by transfection of an antisense oligonucleotide targeting Alu RNA.
Exposure of primary human RPE cells to low oxygen concentrations or increasing doses of cobalt resulted in suppression of DICER1 protein and mRNA. Hypoxia-induced DICER1 suppression coincided with Alu RNA accumulation and NLRP3 inflammasome activation as observed by Caspase-1 cleavage. DICER1 suppression was not rescued by Alu RNA blockade, but hypoxia-induced NLRP3 inflammasome activation and Caspase-1 cleavage was attenuated by Alu RNA blockade. Surprisingly, other hypoxia-induced signaling pathways were also sensitive to Alu RNA blockade. Alu RNA blockade suppressed induction of the glucose metabolism proteins glucose transporter 1 (GLUT1) and GLUT3 and the pro-angiogenic signaling proteins vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2) in hypoxic conditions. Alu RNA did not suppress oxygen-sensitive prolyl hydroxylase domain-containing 2 (PHD2) but did suppress Von Hippel-Lindau (VHL) necessary for degradation of hypoxia-inducible factor-α (HIF-α) transcription factor subunits. Alu RNA-induced VHL reduction coincided with HIF-2α induction.
Hypoxia suppresses DICER1 and causes Alu RNA accumulation. Induction of hypoxia signaling pathways involved in NLRP3 inflammasome activation, glucose metabolism, and angiogenesis require Alu RNA. Alu RNA suppresses VHL and results in HIF-2α stabilization. This work suggests that Alu RNA may be critical for cellular response to hypoxia.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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