Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo.
To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.
Keech
C A
CA
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Gutierrez-Hartmann
A
A
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D006801:Humans; D010902:Pituitary Gland / Q000502:physiology*; D010957:Plasmids; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic
D011388:Prolactin; D004247:DNA; D000242:Cyclic AMP