Redox regulation of thyroid-transcription factors, Pax-8 and TTF-1, is involved in their increased DNA-binding activities by thyrotropin in rat thyroid FRTL-5 cells

Thyroid-enriched transcription factors, Pax-8 and TTF-l, are involved in the thyroid-specific expression of thyroglobulin (TG) gene. Here we demonstrate redox regulation of both factors in vitro and in vivo. When analyzed by electrophoretic mobility shift assay (EMSA), oxidation with diamide abolished the DNA binding of Pax-8. Subsequent reduction with dithiothreitol (DTT) restored the binding. Thioredoxin (TRX), a cellular reducing catalyst, restored the binding more efficiently than DTT. When TTF-l was oxidized with diamide, its binding was decreased and the TTF-1-DNA complex migrated faster on EMSA, DTT reversed these effects, These observations indicate that reduction is required for full DNA binding of Pax-8 and TTF-l in vitro. We then examined whether TSH modulates their binding through redox regulation. Whole cell extracts were prepared from FRTL-5 cells at intervals after TSH treatment without reducing agents and subjected to EMSA. Pax-8 and TTF-1 binding activities were gradually increased during the initial 6 h after TSH. This increase was due to reduction of the factors, since treatment of the extracts with DTT masked the increase by enhancing their binding activities, These results suggest that TSH up-regulates the binding of Pax-8 and TTF-l, at least in part, by reducing the preexisting, oxidized forms in FRTL-5 cells. Northern analysis showed that the increase in TRX mRNA level by TSH in FRTL-5 cells was associated with the increase in the binding activities. Cotransfection of luciferase-reporter plasmid driven by TG promoter with Pax-8- and TRX-expressing plasmids into a heterologous cells revealed that TRX up-regulated the Pax8-mediated TG promoter activity, Taken together, the present study suggests that the redox regulation of Pax-8 and TTF-l by TSH, probably through TRX, modulates the TG gene expression.