September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Hypoxia signaling pathways in retinal pigment epithelium require Alu RNA
Author Affiliations & Notes
  • Charles B Wright
    Ophthalmology and Visual Sciences, University of Kentucky College of Medicine, Lexington, Kentucky, United States
  • Bradley D Gelfand
    Ophthalmology and Visual Sciences, University of Kentucky College of Medicine, Lexington, Kentucky, United States
    Biomedical Engineering, University of Kentucky, Lexington, Kentucky, United States
  • Jayakrishna Ambati
    Ophthalmology and Visual Sciences, University of Kentucky College of Medicine, Lexington, Kentucky, United States
    Physiology, University of Kentucky, Lexington, Kentucky, United States
  • Footnotes
    Commercial Relationships   Charles Wright, None; Bradley Gelfand, None; Jayakrishna Ambati, Allergan (R), Inflammasome Therapeutics (I), iVeena Delivery Systems (I), iVeena Holdings (I), iVeena Pharmaceuticals (I), Olix Pharmaceuticals (F), University of Kentucky (P)
  • Footnotes
    Support  American Heart Association, International Retinal Research Foundation (IRRF), NIH Grants DP1GM114862, R01EY018350, R01EY018836, R01EY020672, R01EY022238, and R01EY024068, Doris Duke Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Ellison Medical Foundation Senior Scholar in Aging Award, Foundation Fighting Blindness Individual Investigator Research Award, Harrington Discovery Institute Scholar-Innovator Award, Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, and Research to Prevent Blindness departmental unrestricted grant
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5813. doi:
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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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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