Oxidation and nuclear localization of thioredoxin‐1 in sparse cell cultures

Abstract

Reactive oxygen species (ROS) were once viewed only as mediators of toxicity, but it is now recognized that they also contribute to redox signaling through oxidation of specific cysteine thiols on regulatory proteins. Cells in sparse cultures have increased ROS relative to confluent cultures, but it is not known whether protein redox states are affected under these conditions. The purpose of the present study was to determine whether culture conditions affect the redox state of thioredoxin-1 (Trx1), the protein responsible for reducing most oxidized proteins in the cytoplasm and nucleus. The results showed that Trx1 was more oxidized in sparse HeLa cell cultures than in confluent cells. The glutathione pool was also more oxidized, demonstrating that both of the major cellular redox regulating systems were affected by culture density. In addition, the total amount of Trx1 protein was lower and the subcellular distribution of Trx1 was different in sparse cells. Trx1 in sparse cultures was predominantly nuclear whereas it was predominantly cytoplasmic in confluent cultures. This localization pattern was not unique to HeLa cells as it was also observed in A549, Cos-1 and HEK293 cells. These findings demonstrate that Trx1 is subject to changes in expression, redox state and subcellular localization with changing culture density, indicating that the redox environments of the cytoplasm and the nucleus are distinct and have different requirements under different culture conditions.

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

“…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. In any case, we observed that PMA induced an increase in TRX-1 non-fully oxidized forms, which was prevented by apocynin.…”

Salmonella Type III Secretion Effector SlrP Is an E3 Ubiquitin Ligase for Mammalian Thioredoxin

“…Another example is the support of peroxiredoxin activity and other antioxidant functions that counteract the triggering of apoptosis through oxidative stress. It is interesting to note that the levels of Trx1 are higher in confluent cultures than in sparse cultures of HeLa cells (38). Other stress response proteins have been shown to increase as cells reach confluence including MnSOD (39) and NADH cytochrome b 5 reductase (40), but the increase of Trx1 seems particularly relevant to the decrease of reactive oxygen species that is also observed in confluent cells (38,40).…”

“…It is interesting to note that the levels of Trx1 are higher in confluent cultures than in sparse cultures of HeLa cells (38). Other stress response proteins have been shown to increase as cells reach confluence including MnSOD (39) and NADH cytochrome b 5 reductase (40), but the increase of Trx1 seems particularly relevant to the decrease of reactive oxygen species that is also observed in confluent cells (38,40). These previous data could give a supportive explanation for our observation that decreased Trx activity in confluent cultures of SlrP expressing cells is correlated with increased cell death (see Fig.…”

Redox Control Systems in the Nucleus: Mechanisms and Functions

“…Nuclear Trx1 is functionally autonomous from cytoplasmic Trx1. A distinct nuclear existence of Trx1 has been demonstrated by immunohistochemical studies, suggesting that Trx1 is imported into the nucleus during oxidative stress (69,169,177,197). Spielberger et al (177) found that cells in sparse culture have increased ROS level, and oxidation of both Trx1 and GSH redox states.…”

“…A distinct nuclear existence of Trx1 has been demonstrated by immunohistochemical studies, suggesting that Trx1 is imported into the nucleus during oxidative stress (69,169,177,197). Spielberger et al (177) found that cells in sparse culture have increased ROS level, and oxidation of both Trx1 and GSH redox states. In sparse cultures, nuclear localization of Trx1 was high relative to high-density cultures, indicating that the redox environments of the cytoplasm and the nucleus are distinct and that Trx1 has an important role of nuclear redox state in cell growth.…”