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 in order to elucidate the roles of these pathways on cell death. Studies herein indicate thiol oxidation contributes to cell death through impaired activity of glutathione dependent (GSH-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 thioredoxin (Trx) systems or glutathione dependent were pharmacologically inhibited with aurothioglucose (ATG) or buthionine sulphoximine 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 a comparatively small pool of proteins rely 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-1 as 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. While 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 of hyperoxic exposure. These data support the hypothesis that hyperoxic impairment of Trx1 negatively impacts HSP90-oxidative responses critical to cell survival with potential implications on pathways implicated in lung development and the pathogenesis of BPD.

Thioredoxin-1/peroxiredoxin-1 as sensors of oxidative stress mediated by NADPH oxidase activity in atherosclerosis

“…In the intracellular compartment, TRX-1 was mainly present as nonfully oxidized forms in basal conditions. In this respect, there have been previous papers showing only the reduced form of TRX-1 in basal conditions [24,[42][43][44][45], while others also ͳͳ showed the two bands observed in the present paper [46,47]. Technical questions and/or culture conditions could account for these potential discrepancies since it has been demonstrated that proliferative status [48] or ion composition [49] could modify TRX-1 oxidative status.…”

A novel mouse model for the identification of thioredoxin-1 protein interactions

“…Therefore, it is not surprising that our data showed novel targets of Trx1 that lie in the nucleus, including ATP-dependent RNA helicase DDX3X (Ddx3x), guanine nucleotide-binding protein subunit beta-2-like-1 (Gnb2l1), heterogeneous nuclear ribonuclear protein A3, and 40S ribosomal protein S3a (Rps3a). Studies have shown that hyperoxia causes Trx1 oxidation [17, 68]. This data indicates that the reactive Cys32 of the flag-hTrx1 C35S under high oxygen conditions could be oxidized, which may explain why fewer targets of Trx1 were identified in this cohort.…”

“…Endogenous expression of a Trx1 protein where the Cys35 residue is mutated to serine creates a stable intermediate between thioredoxin and its substrate and these complexes are then analyzed with mass spectrometry. We have successfully used this technique for proteomic identification of novel Trx1 substrates in lung adenocarcinoma cells [17]. We have taken this method one step further and created a novel transgenic mouse model that expresses the Trx1 substrate trap.…”

“…The prevailing hypothesis of hyperoxia-induced lung injury is that excess oxygen promotes the accumulation of reactive oxygen species and causes oxidation of proteins in several cell survival pathways, which leads to cell death [65, 66]. Our previous work has shown that Trx1 is important for cell survival during hyperoxia [17]. It is well documented that Trx1 translocates to the nucleus during oxidative injury [67] which illustrates the importance of Trx1 in nuclear redox homeostasis.…”

“…It has also been used with plant-derived recombinant thioredoxin in a test tube-based system [15, 16]. Studies from our laboratory have successfully used the Trx1 substrate trap in lung adenocarcinoma cells [17]. Novel nuclear targets, such as the transcription factor PC4 and SFRS1 interaction protein 1 (PSIP1), have been identified by fusing a canonical nuclear localization signal to the substrate trap transgene [18].…”