Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene.

To examine the functional relationship between distinct cis-active elements within the distal enhancer region of the rat PRL gene, we have used deletional and mutational analysis of that region in transient transfection studies in GH3 pituitary tumor cells. Results from these studies demonstrate that the region of the PRL distal enhancer containing the Pit-1-binding sites is critical not only for enhancer activity and the response to cAMP, but also for the response to estradiol. An interaction of the estrogen receptor with factors conferring basal enhancer activity is suggested by studies with a mutant distal enhancer region in which the PRL estrogen response element was converted to a palindromic estrogen response element. To directly examine potential interactions, cotransfection studies using PRL distal enhancer reporter gene constructs and expression vectors for Pit-1 and rat estrogen receptor were performed in two heterologous cell lines. The activity of the reporter gene under the control of the PRL distal enhancer linked to either the thymidine kinase promoter or the PRL proximal promoter was not significantly altered by cotransfection with the Pit-1 expression vector in COS-1 or RAT-1 cells. Coexpression of these reporter constructs and an expression vector for estrogen receptor resulted in only a slight response to estradiol. However, when both Pit-1 and estrogen receptor were cotransfected with the distal enhancer reporter gene, a marked induction was observed in response to estradiol, and this activity was dependent upon the concentration of the Pit-1 expression vector.(ABSTRACT TRUNCATED AT 250 WORDS)