THYROTROPIN-RELEASING-HORMONE ACTION ON THE PROLACTIN PROMOTER IS MEDIATED BY THE POU PROTEIN PIT-1

TRH is known to regulate transcription of the PRL gene in pituitary cells, but little is known about the mechanism involved. We have characterized TRH response elements (TRHREs) in the promoter region of the rat PRL gene and the gene-proximal protein that transmits the TRH signal to these elements. Exposure of GH3 rat pituitary cells to TRH yielded a large specific stimulation of transient expression of a PRL-chloramphenicol acetyltransferase (PRL-CAT) construct containing the PRL promoter region [(-204)PRL-CAT]. Analysis of 5' deletions of this construct implied that regions - 174/-113 and -75/+38 each contain a TRHRE. GH3 cell nuclear extracts are known to footprint four sites, termed, respectively, 1P-4P, on the PRL promoter region. The TRHRE between positions -75/+38 was identified as element 1P, residing at -63/-39, since two copies of a 1P oligodeoxynucleotide transferred a TRH response to either (-39)PRL-CAT or mouse metallothionein-CAT construct (-39)mMT-CAT. Similarly, the more proximal TRHRE may be element 3P, residing at -167/-144, since two copies of this element also transferred a TRH response to (-39)PRL-CAT. Binding of pit-1 to site 1P is known to be capable of activating pituitary cell-specific PRL gene expression. To investigate whether pit-1 can also transduce a TRH signal to this site, oligodeoxynucleotides were prepared corresponding to mutations in either or both of two consensus sequences in site 1P. A strong correlation was observed between the ability of each oligodeoxynucleotide to bind pit-1 and transfer a TRH response to (-39)PRL-CAT, directly implicating pit-1 as the gene-proximal protein that transduces the TRH signal to site 1P. Pit-1 also bound avidly to element 3P (and more weakly to elements 2P and 4P), suggesting that pit-1 may also transduce a TRH signal to element 3P. These results show that the cell-specific activator pit-1 is also a mediator of peptide hormone regulation of the PRL gene.