title: Thioredoxin-1 redox signaling regulates cell survival in response to hyperoxia.
abstract: The most common form of newborn chronic lung disease, bronchopulmonary dysplasia (BPD), is thought to be caused by oxidative disruption of lung morphogenesis, which results in decreased pulmonary vasculature and alveolar simplification. Although cellular redox status is known to regulate cellular proliferation and differentiation, redox-sensitive pathways associated with these processes in developing pulmonary epithelium are unknown. Redox-sensitive pathways are commonly regulated by cysteine thiol modifications. Therefore two thiol oxidoreductase systems, thioredoxin and glutathione, were chosen to elucidate the roles of these pathways on cell death. Studies herein indicate that thiol oxidation contributes to cell death through impaired activity of glutathione-dependent and thioredoxin (Trx) systems and altered signaling through redox-sensitive pathways. Free thiol content decreased by 71% with hyperoxic (95% oxygen) exposure. Increased cell death was observed during oxygen exposure when either the Trx or the glutathione-dependent system was pharmacologically inhibited with aurothioglucose (ATG) or buthionine sulfoximine, respectively. However, inhibition of the Trx system yielded the smallest decrease in free thiol content (1.44% with ATG treatment vs 21.33% with BSO treatment). Although Trx1 protein levels were unchanged, Trx1 function was impaired during hyperoxic treatment as indicated by progressive cysteine oxidation. Overexpression of Trx1 in H1299 cells utilizing an inducible construct increased cell survival during hyperoxia, whereas siRNA knockdown of Trx1 during oxygen treatment reduced cell viability. Overall, this indicated that a comparatively small pool of proteins relies on Trx redox functions to mediate cell survival in hyperoxia, and the protective functions of Trx1 are progressively lost by its oxidative inhibition. To further elucidate the role of Trx1, potential Trx1 redox protein-protein interactions mediating cytoprotection and cell survival pathways were determined by utilizing a substrate trap (mass action trapping) proteomics approach. With this method, known Trx1 targets were detected, including peroxiredoxin-1as well as novel targets, including two HSP90 isoforms (HSP90AA1 and HSP90AB1). Reactive cysteines within the structure of HSP90 are known to modulate its ATPase-dependent chaperone activity through disulfide formation and S-nitrosylation. Whereas HSP90 expression is unchanged at the protein level during hyperoxic exposure, siRNA knockdown significantly increased hyperoxic cell death by 2.5-fold, indicating cellular dependence on HSP90 chaperone functions in response to hyperoxic exposure. These data support the hypothesis that hyperoxic impairment of Trx1 has a negative impact on HSP90-oxidative responses critical to cell survival, with potential implications for pathways implicated in lung development and the pathogenesis of BPD.
authors.affiliation: Basic Biomedical Sciences and The University of South Dakota Sanford School of Medicine, Sioux Falls, SD 57105, USA.
authors.forename: Miranda J
authors.initials: MJ
authors.lastname: Floen
authors.affiliation: Children׳s Health Research Center, Sanford Research, Sioux Falls, SD 57104, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Forred
authors.affiliation: Children׳s Health Research Center, Sanford Research, Sioux Falls, SD 57104, USA.
authors.forename: Elliot J
authors.initials: EJ
authors.lastname: Bloom
authors.affiliation: Department of Pediatrics, The University of South Dakota Sanford School of Medicine, Sioux Falls, SD 57105, USA; Children׳s Health Research Center, Sanford Research, Sioux Falls, SD 57104, USA. Electronic address: peter.vitiello@sanfordhealth.org.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Vitiello
mesh_terms: D001997:Bronchopulmonary Dysplasia / Q000196:embryology / Q000473:pathology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018496:Hyperoxia / Q000378:metabolism*; D008168:Lung / Q000098:blood supply / Q000196:embryology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D054464:Peroxiredoxins / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D015398:Signal Transduction; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D018841:HSP90 Heat-Shock Proteins; D034741:RNA, Small Interfering; C491203:TXN protein, human; D019328:Buthionine Sulfoximine; D013879:Thioredoxins; C515940:PRDX1 protein, human; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase; D005978:Glutathione; D010100:Oxygen
keywords: Cell death; Free radicals; Hyperoxia; Redox signaling; Thioredoxin-1
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