Original contribution
Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts

https://doi.org/10.1016/S0891-5849(01)00617-7Get rights and content

Abstract

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

Introduction

Exposure of mammalian cells to ultraviolet (UV) light induces various deleterious responses [1], [2]. Some of the major harmful effects are DNA damage, systemic immune suppression, and accelerated ageing. The damaging effects of UV-A (320–400 nm) arise from absorption by chromophore, leading to the production of reactive oxygen species (ROS) and the release of cytokines. However, oxidative stress can elicit not only “negative” responses such as cell death but also “positive” responses including cell proliferation and activation [1], [3]. Growing evidence shows that many cellular responses to oxidative stress are triggered by a change in the intracellular redox state [4], which in turn modulates gene expression with strategic activities such as antioxidant metalloenzymes [5].

Cellular redox status is maintained by intracellular redox regulating molecules. The main thiol redox systems developed by cells consist of independent enzymatic couples, the glutathione (γ-glutamyl-cysteinyl-glycine, GSH)/GSH reductase system and the thioredoxin (Trx)/thioredoxin reductase (TR) system [6].

Thioredoxin is a ubiquitous, low molecular weight (Mr 11,000–12,000 Da) redox protein [7], which is reduced by the NADPH-dependent FAD-containing thioredoxin reductase (TR) [8], [9]. Trx has been shown to have several intra- and extracellular functions [10]. Trx was initially reported to be a hydrogen donor for ribonucleotide reductase [11]. Thioredoxin was described as a cytokine-like factor [12] expressed by activated T and B cell lines and involved in cell growth [13]. Trx is mainly located in the cytoplasm and quickly translocates into the nucleus in response to oxidative stress [14]. Locally this protein has been found to modulate the DNA-binding activity of several transcription factors, including NF-κB [15] and heat shock factor [16], and to indirectly modulate AP-1 activity through the intranuclear redox factor ref-1 [17].

Mammalian cells constitutively express Trx. One of its major functions, that of supplying reactive oxygen-scavenging activities, has been demonstrated in vitro and in vivo [18], [19]. Trx has thus been presented as an efficient cell protector and exhibits these properties in a reduced form. Indeed, mutant redox-inactive forms of Trx are unable to protect cells from ROS-induced apoptosis. It was shown that Trx was reduced by TR in a NADPH-dependent reaction. TR was shown to be a dimeric flavoenzyme containing a redox active disulfide and a FAD in each subunit [20]. Mammalian TRs are different from bacterial, plant, and yeast TR because they contain a C-terminal extension with a conserved C-terminal Gly-Cys-Sec-Gly (Sec is selenocysteine) tetrapeptide. The Sec residue in mammalian TR has been involved in the catalytic activity [21].

Until now, GSH has been known to play a key role in protecting human skin cells from UV-induced damage [22], [23], [24]. Nevertheless, it has been reported that the Trx expression increases upon oxidative stress, including UV irradiation in lymphocyte and keratinocyte models [25]. Moreover, TR is highly expressed on the surface of human keratinocytes and melanocytes, suggesting that Trx/TR system provides one of the skin’s first defenses against free radicals generated in response to UV light [26].

The present study analyzes the expression of the Trx/TR system in UVA-irradiated human skin fibroblasts. In order to evaluate the function of Trx, we investigated its antioxidant role against ROS induced by UVA irradiation in Trx-transiently transfected cells. We observed that the redox regulating protein Trx translocates from the cytoplasm to the nucleus and prevents the process of UVA-induced apoptosis.

Section snippets

Materials

RPMI-1640 medium and fetal calf serum (FCS) were purchased from ATGC (ATGC Biotechnologie, Noisy-le-Grand, France). L-glutamine and fungizone were obtained from Roche (Meylan, France), penicillin and streptomycin from Polylabo (Paul Block, Strasbourg, France), and kanamycin and Puck’s saline from Gibco (Grand Island, NY, USA). Phosphate-buffered saline (PBS), containing calcium and magnesium, was purchased from Eurobio (Les Ulis, France). Dihydroethidium was from Bioprobe-Interchim (Montluçon,

Increase in Trx expression of fibroblasts upon UV-irradiation

We first examined the influence of UV radiation on the Trx expression in human skin fibroblasts. Trx is expressed at low level in control cells. Handling of media and exposure to buffer did not modify Trx expression (data not shown). As shown in a representative autoradiography (Fig. 1A), exposure of fibroblasts to UVA (10 J/cm2) resulted in an early (6 h postirradiation) increase in Trx expression with a maximum at 24 h. At 72 h postirradiation, the protein level markedly declined to about

Discussion

Human skin fibroblasts respond to UVA radiation by a highly increased synthesis of the redox protein Trx. In this paper, we show that Trx quickly translocated from the cytoplasm to the nucleus in response to oxidative stress generated by UVA. Using Trx- transfected fibroblasts, we demonstrated that overexpression of Trx was associated with the ability of fibroblasts to survive UV-induced cell death. Trx reduced the level of ROS and prevented the loss of mitochondrial membrane potential elicited

Acknowledgements

This study was made possible by a grant from the Volvic Foundation for Research on Trace Elements. C. Didier was supported by a grant from the French Ligue Contre le Cancer and La Roche-Posay Laboratories, France. We wish to thank Dr. J. Boutonnat, C.H.U. Albert Michallon—France, for technical advice in cytometry. We thank C. Guillermé and F. Labat-Molleur, L. Ydoux, J. Meo, and A. Krawieck for their technical assistance.

References (59)

  • H. Nakamura et al.

    Adult T cell leukemia-derived factor/human thioredoxin protects endothelial F-2 cell injury caused by activated neutrophils or hydrogen peroxide [published erratum appears in Immunol. Lett. 42:213; 1994]

    Immunol. Lett.

    (1994)
  • M. Kaghad et al.

    Genomic cloning of human thioredoxin-encoding genemapping of the transcription start point and analysis of the promoter

    Gene

    (1994)
  • A. Nishiyama et al.

    Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system

    Immunol. Lett.

    (1999)
  • Q.A. Sun et al.

    Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases

    J. Biol. Chem.

    (1999)
  • K.U. Schallreuter et al.

    EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes

    Biochem. Biophys. Res. Commun.

    (1989)
  • K.U. Schallreuter et al.

    Free radical reduction by thioredoxin reductase at the surface of normal and vitiliginous human keratinocytes

    J. Invest. Dermatol.

    (1986)
  • Y. Makino et al.

    Direct association with thioredoxin allows redox regulation of glucocorticoid receptor function

    J. Biol. Chem.

    (1999)
  • M. Bjornstedt et al.

    The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase

    J. Biol. Chem.

    (1994)
  • L.E.S. Netto et al.

    Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties. TSA possesses thiol peroxidase activity

    J. Biol. Chem.

    (1996)
  • M.D. Jacobson

    Reactive oxygen species and programmed cell death

    Trends Biochem. Sci.

    (1996)
  • G. Kroemer et al.

    Mitochondrial control of apoptosis

    Immunol. Today

    (1997)
  • S. Tada-Oikawa et al.

    Role of ultraviolet A-induced oxidative DNA damage in apoptosis via loss of mitochondrial membrane potential and caspase-3 activation

    Biochem. Biophys. Res. Commun.

    (1998)
  • G. Spyrou et al.

    Cloning and expression of a novel mammalian thioredoxin

    J. Biol. Chem.

    (1997)
  • C. Richter et al.

    Control of apoptosis by the cellular ATP level

    FEBS Lett.

    (1996)
  • A. Baker et al.

    Redox control of caspase-3 activity by thioredoxin and other reduced proteins

    Biochem. Biophys. Res. Commun.

    (2000)
  • H.L. Pahl et al.

    Oxygen and the control of gene expression

    Bioessays

    (1994)
  • R.M. Tyrrell

    Activation of mammalian gene expression by the UV component of sunlight—from models to reality

    Bioessays

    (1996)
  • M.A. Stevenson et al.

    X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates

    Cancer Res.

    (1994)
  • H. Nakamura et al.

    Redox regulation of cellular activation

    Annu. Rev. Immunol.

    (1997)
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