Analysis of Pit-1 in regulating mouse TSH beta promoter activity in thyrotropes.
TSH beta 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 TSH beta promoter activity. We demonstrate that both TtT-97 thyrotropic tumors and pituitaries contain four Pit-1 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 alpha TSH cells, a thyrotrope derived cell that no longer expresses TSH beta. Western analysis revealed Pit-1 protein in TtT-97 cells but not in alpha TSH cells. DNase I protection assays localized Pit-1 binding to three areas of the mouse TSH beta promoter. However, basal TSH beta promoter activity was minimally stimulated when alpha TSH cells or TtT-97 thyrotropes were co-transfected with mouse Pit-1 and a mTSH beta luciferase construct. These studies suggest that Pit-1 is not limiting for cell-specific expression of the TSH beta gene in thyrotrope-derived cells and implies that additional thyrotropic factors are likely required.
Gordon
D F
DF
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Haugen
B R
BR
Sarapura
V D
VD
Nelson
A R
AR
Wood
W M
WM
Ridgway
E C
EC
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D013972:Thyrotropin / Q000235:genetics* / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004247:DNA; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D013972:Thyrotropin; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494187:POU1F1 protein, human; C494185:Pou1f1 protein, mouse