MP30-13 FORMATION OF DOUBLE-STRANDED RNA IN HUMAN BLADDER SMOOTH MUSCLE CELLS REGULATES STRESS KINASES AND CELL STRUCTURE IN OXIDATIVE STRESS

Abstract

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2016MP30-13 FORMATION OF DOUBLE-STRANDED RNA IN HUMAN BLADDER SMOOTH MUSCLE CELLS REGULATES STRESS KINASES AND CELL STRUCTURE IN OXIDATIVE STRESS Jing-Hua Yang, Zuohui Zhao, and Kazem Azadzoi Jing-Hua YangJing-Hua Yang More articles by this author , Zuohui ZhaoZuohui Zhao More articles by this author , and Kazem AzadzoiKazem Azadzoi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1245AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Double-stranded RNA (ds-RNA) is RNA with two complementary strands, which is not readily detected in normal cells but regulates ds-RNA-dependent protein kinase (PKR) in cellular stress conditions. Cells exposed to stress develop defensive responses in which different kinases and proteases are highly regulated. Activation of ds-RNA-dependent stress kinase PKR inhibits protein translation via phosphorylation mechanisms. Our goal was to evaluate ds-RNA formation and determine structural consequences of ds-RNA dependent PKR in cultured human bladder smooth muscle cells (SMC) oxidative stress. METHODS Cultured primary human bladder SMC were incubated in normoxia (21% oxygen), continuous hypoxia (2% oxygen) and oxidative stress (2% oxygen for 30 minutes followed by reoxygenation with 21% oxygen for one hour) cycling in this manner for 96 hours in a computerized servo-control cell oxycycler system. Cells were then collected and processed for RT-PCR of ds-RNA precursor Alu-RNA, dot blot analysis of ds-RNA formation, western blotting of ds-RNA-dependent PKR, ELISA of oxidative stress markers, and transmission electron microscopy (TEM). RESULTS Oxidative stress upregulated Alu-RNA expression and led to the formation of ds-RNA in human bladder SMC. Accumulation of ds-RNA resulted in phosphorylation of the stress kinase PKR and produced significant DNA damage. Activation of the ds-RNA/PKR pathway in human bladder SMC was associated with cellular lipid and protein stress markers, cytoskeleton organization and degeneration of the subcellular elements. Ultrastructural consequences of ds-RNA were confirmed by TEM showing disruption of cell structure and widespread organization of subcellular elements. Thickened deformed cell membrane, swollen mitochondria and enlarged ER were found in cells exposed to continuous hypoxia. Markers of cytoskeleton organization characterized by distorted partially lost cell membrane with increased caveolae, enlarged mitochondria with degraded or lost cristae, swollen and splintered ER and increased cytoplasmic lysosomes were evident in cells exposed to oxidative stress. CONCLUSIONS We report the formation of ds-RNA in oxidatively stressed human bladder SMC. Accumulation of ds-RNA and subsequent phosphorylation of PKR activated the ds-RNA/PKR pathway and led to DNA damage, cytoskeleton organization and degeneration of cellular and subcellular elements. Our data imply ultrastructural consequences of ds-RNA and suggest the role of ds-RNA/PKR pathway in human bladder SMC cytoskeleton organization and progressive degeneration under the stress conditions. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e416 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jing-Hua Yang More articles by this author Zuohui Zhao More articles by this author Kazem Azadzoi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...