THYROID Volume 10, Number 4, 2000 Mary Ann Liebert, Inc. Role of Insulin and Serum on Thyrotropin Regulation of Thyroid Transcription Factor-1 and Pax-8 Genes Expression in FRTL-5 Thyroid Cells Diego L. Medina,1·2 Koichi Suzuki,2 Michele Pietrarelli,2 Fumi Okajima,2 Leonard D. Kohn,2 and Pilar Santisteban1 Thyrotropin (TSH), via its cyclic adenosine monophosphate (cAMP) signal, decreases thyrotropin receptor (TSHR) gene expression in FRTL-5 thyroid cells, whereas it increases expression of the thyroglobulin (Tg) gene. Despite the opposite effects of TSH on TSHR and Tg expression, both genes are positively controlled by thy¬ roid transcription factor-1 (TTF-1) and evidence has accumulated that TSH can decrease TTF-1 mRNA levels. In this report, we further characterize the action of TSH on TTF-1 in order to understand its different activities on the TSHR and Tg genes better. The effect of TSH on the TSHR requires the presence of insulin and serum and we show here that also both factors are necessary for the TSH effect to decrease TTF-1 mRNA levels. The decrease is paralleled by a downregulation of TTF-1 protein levels as well as by a decrease in TTF-1 /DNA com¬ plex when the TTF-1 site of the TSHR promoter was used as probe. Again, the decrease requires insulin and serum. The TSH downregulation of TTF-1 mRNA levels is due to a decrease in its transcription rate. Using a luciferase-linked chimera construct spanning 5.18 kb of the TTF-1 5'-flanking region, we show that TSH de¬ creases TTF-1 promoter activity and that this effect depends on insulin and serum. These data contrast with the action of TSH on Tg and Pax-8 gene expression. TSH increases Pax-8 mRNA levels and the increase is ev¬ ident whether insulin and serum are present or not. Moreover, this increase is paralleled by an increase in Pax- 8 protein binding to an oligonucleotide derived from the C site of the Tg promoter, which can bind both TTF- 1 and Pax-8. The present data thus show that TTF-1 gene expression is interdependently regulated by TSH and serum growth factors including insulin. They also show this interdependent-regulation is not duplicated in the case of Pax-8. We suggest that these differences may contribute to the distinct ability of TSH to regulate TSHR versus Tg gene expression in FRLT-5 thyroid cells. Introduction cases, regulation is transcriptional; their promoter activities change in the presence of each of the above hormones and Thyrotropin (TSH) is the main regulator of thyroid fune- growth factors (8-12). The promoters of Tg and TSHR are tion. After binding to the thyrotropin receptor (TSHR), well characterized (13,14). Both minimal promoters contain it initiates a cyclic adenosine monophosphate (cAMP) signal binding sites for thyroid-specific transcription factor-1 (TTF- that finally increases or decreases the expression of the thy- 1) (11,13,15), a homeo-domain containing protein (16). The roid-specific or thyroid-restricted genes that determine the Tg minimal promoter additionally contains binding sites for thyroid phenotype: thyroglobulin (Tg), thyroperoxidase two other thyroid-restricted or thyroid-specific transcription (TPO), TSHR, and the sodium iodide symporter (MS) (1,2). factors, Pax-8 and TTF-2 (13), which are paired- (17) and fork- TSH function can, however, require insulin or insulin-like head-domain (18)-containing proteins, respectively. This is growth factor-I (IGF-I) (3). TSH/cAMP, insulin/IGF-I, or not true for the TSHR (10-12,15) (see Fig. 1 for diagram), serum regulation of expression of genes important for thy- Because the TSHR and Tg genes are hormonally regulated, roid function has been studied extensively in FRTL-5 thy- many studies have attempted to demonstrate the TTF-1, TTF- roid cells (4), particularly for Tg (3,5) and TSHR (6,7). In both 2, and/or Pax-8 mediate transcriptional control by hormones 1Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid, Arturo Duperier 4, Madrid, Spain. ^ell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Insti¬ tutes of Health, Bethesda, Maryland. 295