Mammalian thioredoxin reductase 1 in antioxidant defense, regulation of adipocyte differentiation and as an anticancer drug target

Reactive oxygen species (ROS) are oxygen containing reactive molecules generated as by-products of cellular metabolism. At physiological concentrations, ROS acts as secondary messengers in cellular signaling transduction, but excessive amounts of ROS result in oxidative stress and cellular damage. Several antioxidant enzyme systems include the thioredoxin (Trx)- and glutathione (GSH)-dependent systems together with superoxide dismutases and catalases may act in concert to protect cells and organisms from the toxic effects of excessive ROS. Mammalian thioredoxin reductase 1 (TrxR1), which is a cytosolic selenoprotein with a selenocysteine (Sec, U) residue in a conserved C-terminal GCUG motif, catalyzes the reduction of thioredoxin using NADPH and is known to be involved in antioxidant defense, redox regulation and cell proliferation. This thesis has focused on studying multiple aspects of cellular events and signaling pathways that are modulated by TrxR1.

Paper I. The Sec residue in the C-terminal motif of TrxR1 is highly nucleophilic and can be easily targeted by electrophiles. In this study, we found the mutant p53 activator and anticancer drug lead named APR-246 (PRIMA-1Met) targeted and inhibited both recombinant and cellular TrxR activity. The inhibited TrxR1 still maintained its NADPH oxidase activity, which thus could contribute to the oxidative stress and cell death that are triggered by APR-246. Our findings provide insights into the p53 independent cytotoxicity mechanisms of APR-246 on tumor cells.

Paper II. In this study, we used thiophosphate (SPO3) and selenite to modulate the Sec incorporation into TrxR1 in mammalian cells. We found that SPO3 promoted expression of Sec-to-cysteine substituted forms of TrxR1 and, conversely, selenite increased Sec incorporation in TrxR1. SPO3 treatment also attenuated cisplatin induced toxicity on A549 and HCT116 cells, while selenite supplementation sensitized NIH 3T3 cells to cisplatin but decreased the dependence of these cells on GSH. Taken together, these results show that the selenium status of cells can modulate the cytotoxicity of drugs that target TrxR1 and the glutathione dependence of the cells.

Paper III. Here we utilized Txnrd1 depleted (Txnrd1-/-) mouse embryonic fibroblasts (MEFs) and observed massive cell death upon cultured at low-density in high-glucose medium. The cell death was linked to excessive H2O2 production promoted by high-glucose metabolism. Reconstitution of the cells with Sec-containing TrxR1, but not with the Sec-to-Cys substituted variant, rescued the MEFs from this lethality. These results show that Sec-containing TrxR1 is essential to maintain self-sufficiency of MEFs under high-glucose conditions, due to an essential role in control of glucose-derived H2O2 production. This study is, to our knowledge, the first time identified an essential biological role of Seccontaining TrxR1 that cannot be sustained by the Cys-mutant of the enzyme.

Paper IV. Txnrd1-/- MEFs revealed a strong increase of spontaneous lipogenesis and hormonally induced adipocyte differentiation. The highly promoted adipocyte differentiation capacity was due to unlimited mitotic clonal expansion capacity and dramatically upregulated PPARγ expression. These effects were likely to be connected to increased oxidative signaling in Txnrd1-/- MEFs, because NAC treatment abolished the adipocyte differentiation by blocking mitotic clonal expansion. An increased Akt signaling in Txnrd1-/- MEFs induced by decreased cellular PTEN activity and increased ROS, also contributes to the enhanced adipogenesis. These results suggest that the selenoprotein TrxR1 suppress adipocyte differentiation through inhibition insulin signaling events, mitotic clonal expansion and PPARγ expression.

In summary, this study shows that TrxR1 plays an essential role in antioxidant defense, regulation of adipocyte differentiation and servers as an anticancer drug target.

List of scientific papers

I. Peng X, Zhang MQZ, Conserva F§, Hosny G, Selivanova G, Bykov VJN, Arnér ESJ, Wiman KG. APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase. Cell Death Dis. 2013; (4): e881.
https://doi.org/10.1038/cddis.2013.417

II. Peng X, Xu J, Arnér ESJ. Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase. Biochem J. 2012; 447 (1): 167-74.
https://doi.org/10.1042/BJ20120683

III. Peng X, Mandal PK, Kaminskyy VO, Lindqvist A, Conrad M, Arnér ESJ. Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2. Cell Death Dis. 2014; (5): e1235.
https://doi.org/10.1038/cddis.2014.209

IV. Peng X, Petrus P, Giménez-Cassina A, Conrad M, Rydén M, Arnér ESJ. Thioredoxin reductase 1 suppresses adipocyte differentiation through inhibition of key regulatory signaling events. [Manuscript]