AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1

Abstract

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-kappa B, whose DNA-binding activity is markedly augmented by TRX treatment in vitro. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.

Figure 1

Schematic representation of mutant constructs of TRX used in this experiment.

Figure 2

Effect of transient expression of TRX and/or Ref-1 on AP-1 transactivation in HeLa cells with or without PMA treatment. In each transfection, 1 μg of the pCDSRα-TRX^wild (lanes 2 and 5) or -TRX^C32S/C35S (lanes 3 and 6) expression plasmid and/or 1 μg of the pRc/CMV–Ref-1 (lanes 1–3) expression vector with 1 μg of −73/+63 Col-LUC were used. The total amount of DNA was adjusted to 3 μg with pRc/CMV (Invitrogen). After incubation for 16 hr, PMA (30 ng·ml⁻¹) was added and the cells were kept for 24 hr. The cells were harvested after 40 hr, and luciferase activity was determined. The results are the means ± SE of three experiments (each in duplicate) and presented as fold increases in luciferase activity over the baseline seen in lane 4 without PMA treatment.

Figure 3

Effect of PMA treatment on subcellular distribution of TRX and Ref-1 in HeLa cells. Indirect immunofluorescence labelling, using anti-TRX antibody (A–C) or anti-Ref-1 antibody (D). (A) Before PMA treatment. (B) After 1-hr treatment. (C and D) After 24-hr treatment.

Figure 4

Evidence that TRX associated with Ref-1 through the redox-active cysteines in vitro. Various TRX mutants containing cysteine-to-serine or cysteine-to-alanine substitutions were cross-linked with Ref-1 or wbFos by diamide. After incubation, the complexes were resolved by electrophoresis under reducing or oxidizing conditions and detected by antibodies. (A) Western blot analysis of various TRX and Ref-1 complex by use of anti-Ref-1 antibody. (B) Western blot analysis of TRX^{C32S/C69S/C73S} and Ref-1 complex by use of anti-TRX antibody. (C) Western blot analysis of TRX^{C32S/C69S/C73} and wbFos by use of anti-c-Fos antibody or anti-TRX antibody.

Figure 5

Demonstration of a physical association between TRX and Ref-1 by mammalian two-hybrid assay in COS-7 cells. (A) An association between wild-type TRX and Ref-1 was monitored by a luciferase assay. (B) An association between TRX mutants and Ref-1 was monitored by a luciferase assay. The relative fold induction of luciferase activity was calculated by normalizing to the β-galactosidase activity. The results are the means ± SE of three experiments (each in duplicate) and presented as fold increases in luciferase activity over the baseline seen in lane 1.