The Role of Adenosine Deaminase Acting on RNA (ADAR) Family of Proteins in Hyperoxia Induced Acute Lung Injury

Abstract

IntroductionRNA editing is a post‐transcriptional modification catalyzed by the ADAR family of proteins. Adar1 plays an important role in the innate immune response, organ development, and homeostasis. Adar1 isoforms (interferon stimulated p150 and constitutive p110) and Adarb1 are highly expressed in lungs. Hyperoxia impairs the lung's innate immune response, but the role of Adars in the hyperoxia‐induced acute lung injury (HALI) model is unknown. Therefore, the role of Adar1, Adarb1, and aminoacyl tRNA synthase complex‐interacting multifunctional protein 2 (AIMP‐2) in HALI (using C57BL/6 and Adarb1 transgenic mice) were investigated.MethodsC57BL/6 mice or Adarb1 transgenic mice were exposed to 95 % oxygen for 48h. Lungs were perfused and tissue harvested for RNA, protein extraction, and histology. Sections were stained with H&E and Masson Trichome. Adar1 Alexa 594, AIMP2, and pro‐SPC were used for Immunofluorescence (IF) analysis. Total RNA was extracted, cDNA synthesized, and qRT‐PCR performed using specific primers and the SYBR green kit (Biorad). Relative fold change was calculated using ΔΔCt method using 18S as a calibrator. Lung tissues were fractionated. Nuclear and cytoplasmic extracts were analyzed by Western blotting.ResultsAdar1 and Adarb1 transcripts in lungs remain unchanged in mice exposed to 48h of hyperoxia versus normoxia. However, Adar1 (p150 and p110) expression in cytoplasmic and nuclear fractions significantly decreases under hyperoxia versus normoxia. Adarb1 levels remain unchanged under hyperoxia. Adar1 signals decrease in lung sections of mice exposed to 48h of hyperoxia. H & E stained lung sections of Adarb1 transgene and control mice exposed to 48h of hyperoxia show alveolar disorganization, alveolar thickening, and immune cell infiltration. Masson trichome staining of Adarb1 transgenic mice exposed to hyperoxia show collagen staining in alveolar regions similar to hyperoxic controls. Real time PCR of pro‐apoptotic Bax1 demonstrates a significant increase in both the Adarb1 transgene and wild type control exposed to hyperoxia relative to normoxic controls. AIMP2 mRNA levels increase significantly in wild type mice exposed to hyperoxia relative to normoxic controls, without an increase in protein levels. Similar results were obtained by IF analysis of AIMP2 in lung sections of hyperoxia and normoxia mice.ConclusionsThe isoforms of Adar1, p150 and p110, may play a significant role in maintaining lung homeostasis. Overexpression of Adarb1 in transgenic mice does not protect against HALI. AIMP2 does not play a role in regulating Adar1 or Adar2 stability under hyperoxic conditions.Support or Funding InformationThis work is supported by NIH RO1 grant (HL105932) to N.K.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.