DETERMINANTS FOR THE REPRESSION OF ESTROGEN-RECEPTOR TRANSCRIPTIONAL ACTIVITY BY LIGAND-OCCUPIED PROGESTIN RECEPTORS

There is considerable evidence for cross-talk between the estrogen and progestin signaling pathways, including examples of repression or att enuation of estrogen-stimulated endpoints by progestin receptor (PR) a gonists and antagonists. We have previously described an experimental system for examining aspects of this cross-talk, namely the repression of estrogen receptor (ER) transcriptional activity by liganded PR (Kr aus, W. L., Weis, K. E., Katzenellenbogen, B. S., Mol. Cell. Biol. 15 (1995) 1847-1857). Under promoter and cell type conditions where ligan ded PR was not a good activator of transcription, PR isoforms were sho wn to act as potent ligand-dependent repressors of ER transcriptional activity. In the current study, we have identified multiple determinan ts of this repression by systematically manipulating potentially impor tant variables in this system (e.g. PR A:PR B ratio, sequence of the r esponse elements, receptor structure, and ligand type). Alterations in several of these parameters had profound effects on the ability of PR to repress the activity of ER. Decreases in the PR A:PR B ratio and c hanges in the sequence of the progestin response element in the report er gene construct abolished the repressive action of agonist-occupied PR A on ER transcriptional activity. In addition, point or deletion mu tations in the amino-terminal A/B region of ER, including a triple poi nt mutation which eliminates phosphorylation sites previously shown to be important in the activity of the receptor, made the ER more sensit ive to the repressive actions of liganded PR. The PR ligands that prom oted the most potent repression of ER activity were those with 11 beta phenyl substitutions, suggesting that the phenyl moiety in the 11 bet a position is the important structural feature leading to strong repre ssion. Interestingly, changes in the structure of the ER ligand and th e sequence of the estrogen response element did not influence the magn itude of repression by PR. The fact that alterations in these check po ints along the estrogen signaling pathway had little or no effect on t he magnitude of repression suggests that liganded PR interferes with t he ability of ER to interact productively with the transcriptional mac hinery; in other words, PR-mediated repression occurs downstream of th e events leading to the ligand-dependent conversion of ER to a transcr iptionally active form. Our results indicate that a number of paramete rs which are naturally varied in vivo, such as the sequence of PR DNA binding sites and the PR A:PR B ratio, can dramatically alter the repr ession of ER activity by liganded PR, and may explain the differential affects of progestin-occupied PR on the expression of different estro gen regulated genes. (C) 1997 Elsevier Science Ltd. All rights reserve d.