Hiroshi Sono

Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D3 up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1α,25-dihydroxyvitamin D3 caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

Transactivation of an inducible anti-oxidative stress protein, human thioredoxin by HTLV-I Tax.

Adult T-cell leukemia derived factor (ADF)/human thioredoxin (TRX), which has thiol reducing and radical scavenging activities, plays an essential role on cellular protection against oxidative stress and cell death. TRX itself is induced by various oxidative stress as well as the Human T-cell lymphotropic virus type I (HTLV-I) Tax protein. To investigate the mechanism of this induction, the promoter region of the TRX gene was analyzed. Chloramphenicol acetyltransferase (CAT) reporter constructs containing the TRX promoter sequences responded to the overexpression of the Tax protein, whereas various oxidative agents activated the TRX promoter through a newly identified oxidative responsive element. Moreover, TRX was translocated from the cytoplasm into the nucleus by ultraviolet irradiation, suggesting its possible role on sensing and transducing oxidative signals.

Thioredoxin-mediated redox control of human T cell lymphotropic virus type I (HTLV-I) gene expression.

Thioredoxin (TRX) is a small ubiquitous protein with multiple biological functions, including the thiol-mediated redox-regulation of gene expression. We have previously demonstrated that human TRX is overexpressed as a major protein oxidoreductase in human T cell lymphotropic virus type I (HTLV-I)-infected cells. In the present study, we investigated the relationship between TRX and viral gene expression in HTLV-I infection. To study the mechanism that causes overexpression of TRX in HTLV-I-infected cells, we first examined the effect of the HTLV-I transactivator, Tax, on TRX expression. Induction of HTLV-I Tax protein increased the expression of TRX protein in a Tax-transfected Jurkat cell line, JPX-9. Moreover, chloramphenicol acetyltransferase (CAT) analysis with a reporter gene containing the TRX promoter revealed that Tax activates the transcription of TRX gene. To study the role of overexpressed TRX in HTLV-I infection, we next examined the effect of TRX on HTLV-I long terminal repeat (LTR)-mediated transcription using CAT analysis. In an HTLV-I-infected human T cell line MT-2, the HTLV-I LTR transactivation was suppressed by the overexpression of wild-type TRX, but activated by the introduction of inactive mutant TRX. Moreover, in HTLV-I negative Jurkat T cells, the HTLV-I LTR transactivation induced by Tax was also repressed by overexpression of wild-type TRX. Because cellular redox changes were shown to affect the HTLV-I gene expression, it is likely that TRX modulates the HTLV-I gene expression by regulating cellular redox state. Taken together, these findings suggest that overexpressed TRX, which is induced by HTLV-I Tax, may play an important role in HTLV-I infection through the negative regulation of viral gene expression.

Exogenous PML/RARα Fusion Gene Responds to All-trans Retinoic Acid Results in Differentiation of the Human B Cell Line.

The interaction of an exogenous PML/RARα fusion gene, associated with acute promyelocytic leukemia, with all-trans retinoic acid (ATRA) was examined in B-lymphoid cell lines. RPMI8866 cells were transfected with PML/RARα cDNA in the expression vector pGD and two stable transformants (RPMI8866Y-4 and RPMI8866Y-17) were established by selection with G418. ATRA inhibited the growth of those stable transformants, as assessed by [3H]-thymidine incorporation, but had no effect on the growth of control cells stably transformed with neomycin resistant gene alone. ATRA also increased expression of CD38 and immunoglobulin production in RPMI8866Y-4 cells but not in control cells. When these results are taken together, it can be observed that the exogenous PML/RARα fusion gene responds to ATRA, which results in cell differentiation of the human B cell line.