PIT-1 BINDING SEQUENCES PERMIT CALCIUM REGULATION OF HUMAN PROLACTIN GENE-EXPRESSION

This study examines the regulation of the human PRL (hPRL) gene promoter by intracellular calcium. Deletants of the 5'-flanking region of the hPRL gene and constructs consisting of the thymidine kinase promoter linked to the first or second proximal Pit-1 binding site were fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. With the complete 5-kilobase pair (kbp) hPRL promoter sequence the calcium channel agonist Bay K8644 induced a significant 2-fold increase in CAT reporter gene expression and the antagonist verapamil a 4.5-fold reduction, using GH3 cells cultured in physiological levels of calcium. The transcriptional response to calcium influx was similar with a series of 5'-deleted hPRL-CAT constructs including those that comprised the proximal (up to 740 bp) or distal (-1300- to -1700-bp) sequences alone. When treating cells cultured in low calcium conditions the induction with the hPRL promoter increased to 5-fold on the addition of exogenous calcium and Bay K8644. The pituitary-specific expression of the hPRL gene is conferred by the interaction of the pituitary-specific factor Pit-1 with several binding sites located in the 5'-flanking DNA, of which three are located in the proximal region. This suggested that Pit-1 binding sites may be involved in the calcium response. The presence of either the first or the second proximal Pit-1 binding site alone linked to a thymidine kinase promoter was sufficient to confer a significant 2-fold increase in reporter gene response to exogenous calcium and Bay K8644, while the constructs containing multiple Pit-1 sites linked to a thymidine kinase promoter conferred a further increase. This transcriptional response was lost when the constructs were transfected into the (calcium-responsive) nonpituitary HIT-T15 cell line. The calcium response was also lost when point mutations were introduced into a Pit-1 binding site. Our data indicate that the hPRL gene promoter is responsive to raised intracellular calcium. This response involves Pit-1 binding sites and appears to require the presence of Pit-1 protein.