Interactions of thyrotropin-releasing hormone, phorbol ester, and forskolin-sensitive regions of the rat thyrotropin-beta gene.
Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
Shupnik
M A
MA
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22903.
Rosenzweig
B A
BA
Showers
M O
MO
D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D005796:Genes / Q000187:drug effects*; D005822:Genetic Vectors; D008297:Male; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000494:pharmacology; D051381:Rats; D013972:Thyrotropin / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D010703:Phorbol Esters; D005576:Colforsin; D013973:Thyrotropin-Releasing Hormone; D013972:Thyrotropin; D000242:Cyclic AMP; D011493:Protein Kinase C