8 - The Thioredoxin Reductase Inhibitor Auranofin Induces Heme Oxygenase-1 in Lung Epithelial Cells Via Nrf2-dependent Mechanisms

Abstract

Thioredoxin reductase-1 (TrxR1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and attenuates lung injury in a murine bronchopulmonary dysplasia (BPD) model. Heme oxygenase-1 (HO-1) is regulated by Nrf2, although not exclusively. We have observed disproportionate increases in HO-1 levels in vivo and in vitro following TrxR1 inhibition; however, the role of Nrf2 in HO-1 induction by TrxR1 inhibitors is unclear. To test the hypothesis that TrxR1 inhibition induces HO-1 via Nrf2-dependent mechanisms, Nrf2 knockout (KO) murine transformed club cells (mtCCs) were generated using CRISPR/Cas9 and Nrf2 deletion confirmed by qRT-PCR and western blot. Cells were treated with 0.5 µM of the TrxR1 inhibitor, auranofin (AFN), or control (DMSO) for 3h and lysates were collected. TrxR1 activity was determined by insulin disulfide reductase assay. HO-1 mRNA and protein expression were determined by qRT-PCR and western blot, respectively. Data (n=5-6) were analyzed by 2-way ANOVA with Tukey’s post hoc (p In KO mtCCs, baseline TrxR1 activity was 50% lower than that in wild type (WT) cells. AFN treatment decreased TrxR1 activity by 50% compared to respective control-treated WT and KO cells. In AFN-treated WT mtCCs, Hmox1 levels were 30-fold greater than in control cells. This corresponded with a 75-fold increase in HO-1 protein expression. In AFN-treated KO cells, we detected only a 2-fold increase in Hmox1; however, no increase in HO-1 protein was detected. To confirm that the KO mtCCs are able to generate Hmox1 and HO-1, WT and KO mtCCs were treated with hemin (5 µM), a Nrf2-independent HO-1 inducer, or DMSO control for 8h. In hemin-treated mtCCs, Hmox1 levels were increased 5-fold in both WT and KO mtCCs when compared to their respective controls. Compared to their respective controls, HO-1 protein levels were increased 15-fold in WT and 5-fold in KO cells. Our data suggest that Nrf2 is the primary mechanism by which AFN increases HO-1 in mtCCs. HO-1 has been identified as a potential therapeutic target to prevent BPD. Future studies will investigate the role of Nrf2 in HO-1 induction in the lung in vivo and the contribution of HO-1 to the protective effects of TrxR1 inhibitors in our BPD model.