ANALYSIS OF PIT-1 IN REGULATING MOUSE TSH-BETA PROMOTER ACTIVITY IN THYROTROPES

TSHbeta gene expression is restricted to pituitary thyrotropes. Since Pit-1 is present in these cells, we characterized Pit-1 RNA and protein in thyrotropes, and tested its function in regulating TSHbeta promoter activity. We demonstrate that both TtT-97 thyrotropic tumors and pituitaries contain four Pit-I transcripts of 3.2, 2.6, 2.4, and 1.9 kb, respectively. Only two transcripts of 2.7 and 2.1 kb were detected in alphaTSH cells, a thyrotrope derived cell that no longer expresses TSHbeta. Western analysis revealed Pit-I protein in TtT-97 cells but not in alphaTSH cells. DNase I protection assays localized Pit-1 binding to three areas of the mouse TSHbeta promoter. However, basal TSHbeta promoter activity was minimally stimulated when alphaTSH cells or TtT-97 thyrotropes were co-transfected with mouse Pit-I and a mTSHbeta luciferase construct. These studies suggest that Pit-I is not limiting for cell-specific expression of the TSHbeta gene in thyrotrope-derived cells and implies that additional thyrotropic factors are likely required.