PITUITARY-TYPE TRANSCRIPTION OF THE HUMAN PROLACTIN GENE IN THE ABSENCE OF PIT-1

We describe a human (h) PRL-producing cell line, SKUT-1B-20, which we isolated as a subclone of a uterine sarcoma cell line. Although this c ell line is of uterine origin, it does not use the decidual-specific u pstream promoter of the hPRL gene, but transcribes the hPRL gene from the downstream pituitary-type transcription start site, as determined by Northern blot, reverse transcriptase-polymerase chain reaction and primer extension analyses. This is particularly intriguing because SKU T-1B-20 cells lack the transcription factor Pit-1. No Pit-1 messenger RNA was detectable by reverse transcriptase-polymerase chain reaction, and endogenous Pit-1 target genes (GH, PRL, and Pit-1) were refractor y to transfected Pit-1 expression vector, whereas in cotransfection ex periments, Pit-1 efficiently activated reporter gene fusion constructs carrying 5'-flanking sequences of the human and rat PRL or the mouse Pit-1 genes. By transfecting reporter genes containing 8.7 kilobases o f DNA flanking the hPRL pituitary-specific start site (hPRL-8700/Luc) and deletions thereof, we located a Pit-I-independent cis-active regio n more than 7 kilobases upstream of the start site. The most distal 16 50 or 880 base pairs of the hPRL genomic fragment (which extends to -8 784 base pairs), when placed directly upstream of the homologous hPRL or the heterologous thymidine kinase promoters, conferred transcriptio nal activation to those promoters. SKUT-1B-20 cell-specific activation of hPRL-8700/Luc could not be suppressed by the introduction of an in hibitor of protein kinase A (PKA), PKI. This is the first demonstratio n of pituitary-type PRL gene transcription independent of Pit-1 and ac tivation of the PKA pathway. The SKUT-1B-20 cell line was then used in reconstitution experiments to delineate the role of Pit-1 in modulati ng the transcriptional effects of phorbol ester, PKA, and estrogen rec eptor (ER) on the hPRL gene. The low response of hPRUL/luciferase fusi on genes to phorbol ester was greatly enhanced by cotransfected Pit-1 and was mediated by the proximal region between -250 and -38. The cata lytic subunit of PKA, CP, was able to elicit a moderate induction of h PRL-8700/Luc even in the absence of Pit-1; the response was strongly a mplified by coexpression of Pit-1. A potential estrogen response eleme nt has been located in the hPRL gene sequence at a position similar to that of the estrogen response element of the rat PRL gene immediately adjacent to the distal enhancer. in striking contrast to the dramatic cooperative action of ER and Pit-1 on the rat PRL gene, which has bee n reported previously and was also supported by SKUT-1B-20 cells, no s uch response was obtained with hPRL gene constructs. Liganded ER cause d a mere P-fold induction of reporter gene activity regardless of the absence or presence of Pit-1. This demonstration of a functional dissi milarity between rat and hPRL gene regulatory regions is congruous wit h the different roles of estrogen in lactotrope control in the two spe cies.