THE COMBINATION OF PIT-1 AND PIT-1T HAVE A SYNERGISTIC STIMULATORY EFFECT ON THE THYROTROPIN BETA-SUBUNIT PROMOTER BUT NOT THE GROWTH-HORMONE OR PROLACTIN PROMOTERS

Pit-1 is a pituitary-specific transcription factor with protein expression limited to thyrotrope, somatotrope, and lactotrope cells of the anterior pituitary gland. We have recently described a thyrotrope-specific variant isoform of Pit-1, called Pit-1T, which contains an additional 14 amino acids in the activation domain generated by on alternate 3'-splicing choice. Pit-1T, in the presence of Pit-1, stimulates the thyrotropin beta-subunit (TSH beta) promoter in a thyrotrope-derived cell that lacks all Pit-1 isoform proteins. Three laboratories have identified another Pit-1 splice variant, called Pit-1 beta, which contains an additional 26 amino acids in the activation domain that is generated by a similar 3'-alternate splice choice. Pit-1 beta has been shown to stimulate the GH promoter, but not the PRL or TSH beta promoters. In this report, we evaluate the effect of the three Pit-1 isoforms (Pit-1, Pit-1T, and Pit-1 beta) on the GH, PRL, and TSH beta promoters when introduced into different cell types. The combination of Pit-1 and Pit-1T had a synergistic stimulatory effect on the TSH beta promoter, but not on the PRL or GH promoters in a thyrotrope-derived cell line that lacks all Pit-1 protein isoforms (alpha TSH cells). When added to GH3 cells, which lack only the Pit-1T isoform, Pit-1T selectively stimulated the TSH beta promoter and not the GH or PRL promoters, suggesting that the thyrotrope-specific Pit-1T exhibits a promoter-specific effect. Basal TSH beta promoter activity in thyrotropes was lost with a mutation of the -133 to -100 region. Deletional analysis of the TSH beta promoter in GH3 cells and TtT-97 thyrotropes showed that Pit-1T mediated TSH beta promoter stimulation through two different regions (-255 to -222 and -133 to -100). However, a mutation of the -133 to -100 region abolished Pit-1T-mediated stimulation in TtT-97 thyrotroptes but not GH3 somatomammotropes. These data suggest that the -133 to -100 region of the TSH beta promoter is important for thyrotrope-specific expression as well as Pit-1T-mediated stimulation in thyrotropes.