Identification of a cis-regulatory element and a thyroid-specific nuclear factor mediating the hormonal regulation of...

The mechanism for hormonal regulation of rat thyroperoxidase (rTPO) gene transcription in rat FRTL-5 thyroid cells has been investigated. Transient transfection experiments demonstrate that the minimal rTPO promoter that confers thyroid-specific expression also confers responsiveness to TSH and insulin. TSH induces a 7-fold increase in promoter activity, and the induction is detected almost immediately after the addition of the hormone. Insulin also stimulates TPO promoter activity, but the effect of this hormone is weaker and slower than that of TSH. The effect of TSH in increasing TPO promoter activity is mimicked by the cAMP agonist forskolin. The calcium-protein kinase C pathway is also involved in the regulation of the rTPO promoter activity, since a calcium ionophore (A23187) and phorbol esters [12-O-tetradecanoyl-phorbol-13-acetate (TPA)] inhibit it quickly. These data indicate that the region of the rTPO promoter used here contains the DNA signals necessary for its hormonal regulation. Protein-DNA binding studies show that the thyroid-specific nuclear protein TTF-2, which binds to the rTPO promoter, is induced by TSH and forskolin, and this effect is clearly observable as early as 5 h post induction. Moreover, the DNA binding activity of TTF-2 is inhibited by both A23187 and TPA. Heterologous promoter constructs containing four, eight, or 12 tandem repeats of an oligonucleotide that includes the TTF-2 binding site increase their activity in response to TSH, forskolin, and insulin, while the the presence of A23187 or TPA inhibits their activity. These data indicate that the TTF-2 protein plays an important role in the hormonal control of thyroid-specific transcription.