Selenium supplementation of lung epithelial cells enhances nuclear factor E2-related factor 2 (Nrf2) activation following thioredoxin reductase inhibition

Abstract

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.

Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Graphical abstract

Fig. 1

TXNRD1 activity in mtCC lysates. mtCCs were cultured in 10% or 5% fetal bovine serum (FBS) supplemented with 0, 10, 25, or 100 nM Na2SeO3 for 72 h. TXNRD1 activity (ng/mg lysate) was determined by insulin-disulfide reductase assay as described in Methods. Data (mean±SEM, n = 6) were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. (*p < 0.033 vs 10% FBS; #p < 0.038 vs 5% FBS + 0 nM Na2SeO3; $p < 0.034 vs 5% FBS + 10 nM Na2SeO3).

Fig. 2

Effects of Se supplementation on expression and activity of Sec-containing enzymes. mtCCs were cultured for 72 h in 5% FBS-containing media supplemented with 0, 10, 25 or 100 nM Na2SeO3. (A) TXNRD1 expression; (B) TXNRD1 activity; (C) GPX2 expression; and, (D) GPx activity. Linear regression analysis revealed a positive association between Na2SeO3 and GPx activity. Data (expressed as fold change vs 0 Na2SeO3, mean±SEM, n = 5–9) were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. (*p < 0.011 vs 0 nM Na2SeO3; @p < 0.014 vs 10 nM Na2SeO3; %p = 0.0458 vs 25 nM Na2SeO3;$p = 0.042, R2 = 0.92).

Fig. 3

Expression and activity of Sec-containing enzymes in the presence of AFN. mtCCs were cultured in 5% FBS-containing media supplemented with 0, 10, 25 or 100 nM Na2SeO3 for 72 h, treated with 0.5 µM AFN for 1 h, and lysates were collected. (A) TXNRD1 expression; (B) TXNRD1 activity; (C) GPX2 expression; and, (D) GPx activity. Data (expressed as fold change vs 0 Na2SeO3 no AFN control, mean±SEM, n = 5–9) were analyzed by one-way ANOVA followed by Tukey's post hoc analysis. (*p < 0.011 vs 0 nM Na2SeO3 no AFN; $p < 0.031 vs 0 nM Na2SeO3+AFN; @p = 0.02 vs 10 nM Na2SeO3 + AFN; %p = 0.02 vs 25 nM Na2SeO3 + AFN).

Fig. 4

Effects of AFN on TXNRD1 activity 3 h after treatment. mtCCs were cultured in 5% FBS-containing media supplemented with 0, 10, 25 or 100 nM Na2SeO3 for 72 h. Cells were then treated with 0.5 µM AFN or vehicle for 1 h, washed, placed in fresh media, and lysates collected 2 h later. Data were analyzed (mean±SEM, n = 3–5) by one-way ANOVA followed by Tukey's post hoc analysis. (*p < 0.041 vs 0 nM control; #p < 0.012 vs respective [Na2SeO3]; $p = 0.0003 vs 0 nM Na2SeO3 + AFN).

Fig. 5

Nuclear Nrf2 expression and ARE-luciferase activity. mtCC were cultured in 5% FBS-containing media containing 0, 10, 25 or 100 nM Na2SeO3. (A) Cells were cultured in the presence or absence of 0.5 µM AFN for 1 h and Nrf2 expression was determined by western blotting of nuclear fractions. (B, C) mtCCs were cultured as above, transfected with ARE-luciferase and Renilla luciferase plasmid DNA for 24 h, and incubated in the presence or absence of 0.5 µM AFN for 18 h. (B) Luciferase activity in 0, 10, 25 or 100 nM Na2SeO3-supplemented cells. (C) Fold-change luciferase activity in control and AFN-treated cells. Data (mean±SEM, n = 3) were analyzed by one-way ANOVA followed by Tukey's post hoc analysis (A, B) or t-test (C). (*p < 0.05 vs respective [Na2SeO3]; $p < 0.03 vs 0 nM Na2SeO3+AFN; #p < 0.02 vs 10 mM Na2SeO3+AFN).

Fig. 6

NQO1 and HO-1 mRNA levels. mtCCs were cultured in 5% FBS-containing media supplemented with 0, 10, 25 or 100 nM Na2SeO3 for 72 h. (A) Nqo1 and Hmox1 mRNA levels after 72 h incubation. Cells were cultured in the presence or absence of 0.5 µM AFN for 1 h, media was changed, and lysates collected 2 h later. washing and incubation for an additional 2 h. (B) Nqo1 and Hmox1 mRNA levels in control and AFN-treated cells. Data (expressed as fold change vs control, mean±SEM, n = 6) were analyzed by t-test. (p < 0.05 vs respective [Na2SeO3] control).