This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C 6 -ceramide, but not by the biologically inactive analog dihydro-C 6 -ceramide, in a time-and dose-dependent manner, with 85% inhibition produced by 20 M showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 M for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C 6 -ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.

The trace element selenium (Se) contributes to redox signaling, antioxidant defense, and immune responses in critically ill neonatal and adult patients. Se is required for the synthesis and function of selenoenzymes including thioredoxin (Trx) reductase-1 (TXNRD1) and glutathione peroxidases (GPx). We have previously identified TXNRD1, primarily expressed by airway epithelia, as a promising therapeutic target to prevent lung injury, likely via nuclear factor E2-related factor 2 (Nrf2)-dependent mechanisms. The present studies utilized the TXNRD1 inhibitor auranofin (AFN) to test the hypothesis that Se positively influences Nrf2 activation and selenoenzyme responses in lung epithelial cells. Murine transformed Club cells (mtCCs) were supplemented with 0, 10, 25, or 100 nM Na2SeO3 to create a range of Se conditions and were cultured in the presence or absence of 0.5 μM AFN. TXNRD1 and GPX2 protein expression and enzymatic activity were significantly greater upon Se supplementation (p < 0.05). AFN treatment (0.5 μM AFN for 1 h) significantly inhibited TXNRD1 but not GPx activity (p < 0.001). Recovery of TXNRD1 activity following AFN treatment was significantly enhanced by Se supplementation (p < 0.041). Finally, AFN-induced Nrf2 transcriptional activation was significantly greater in mtCCs supplemented in 25 or 100 nM Na2SeO3 when compared to non-supplemented controls (p < 0.05). Our novel studies indicate that Se levels positively influence Nrf2 activation and selenoenzyme responses following TXNRD1 inhibition. These data suggest that Se status significantly influences physiologic responses to TXNRD1 inhibitors. In conclusion, correction of clinical Se deficiency, if present, will be necessary for optimal therapeutic effectiveness of TXNRD1 inhibitors in the prevention of lung disease.

Background Aurothioglucose- (ATG-) mediated inhibition of thioredoxin reductase-1 (TXNRD1) improves alveolarization in experimental murine bronchopulmonary dysplasia (BPD). Glutathione (GSH) mediates susceptibility to neonatal and adult oxidative lung injury. We have previously shown that ATG attenuates hyperoxic lung injury and enhances glutathione- (GSH-) dependent antioxidant defenses in adult mice. Hypothesis The present studies evaluated the effects of TXNRD1 inhibition on GSH-dependent antioxidant defenses in newborn mice in vivo and lung epithelia in vitro. Methods Newborn mice received intraperitoneal ATG or saline prior to room air or 85% hyperoxia exposure. Glutamate-cysteine ligase (GCL) catalytic (Gclc) and modifier (Gclm) mRNA levels, total GSH levels, total GSH peroxidase (GPx) activity, and Gpx2 expression were determined in lung homogenates. In vitro, murine transformed club cells (mtCCs) were treated with the TXNRD1 inhibitor auranofin (AFN) or vehicle in the presence or absence of the GCL inhibitor buthionine sulfoximine (BSO). Results In vivo, ATG enhanced hyperoxia-induced increases in Gclc mRNA levels, total GSH contents, and GPx activity. In vitro, AFN increased Gclm mRNA levels, intracellular and extracellular GSH levels, and GPx activity. BSO prevented AFN-induced increases in GSH levels. Conclusions Our data are consistent with a model in which TXNRD1 inhibition augments hyperoxia-induced GSH-dependent antioxidant responses in neonatal mice. Discrepancies between in vivo and in vitro results highlight the need for methodologies that permit accurate assessments of the GSH system at the single-cell level.