DIFFERENTIAL REGULATION OF HUMAN PROGESTERONE RECEPTOR-A AND RECEPTOR-B FORM-MEDIATED TRANSACTIVATION BY PHOSPHORYLATION

Hormone-dependent phosphorylation of progesterone receptors (PRs) play s a functional role in their transcriptional activity. However, hormon e-independent phosphorylation has also been shown to modulate the chic ken PR-mediated trans-activation in the presence of phosphorylating ag ents. The present study was designed to investigate the effects of pro tein kinase A- and protein kinase C-mediated signal transduction pathw ays on the regulation of the activity of the two forms of human PR (hP R(A) and hPR(B)). Similar to chicken PR, hPR was activated by 8-bromo- cAMP (8-Br-cAMP) in the absence of ligand, whereas 8-Br-cAMP synergize d with the progestin agonist R5020 to amplify hPR(A)- and hPR(B)-media ted reporter activity. Interestingly, the effect of 8-Br-cAMP was much more pronounced on hPR(A)-induced trans-activation than on hPR(B). Th is differential regulation by 8-Br-cAMP could also be mimicked by okad aic acid. Both mouse mammary tumor virus-thymidine kinase-chlorampheni col acetyl transferase and progesterone response element-thymidine kin ase-chloramphenicol acetyl transferase showed a similar response to 8- Br-cAMP in the presence of R5020. Protein kinase C, on the other hand, did not discriminate between hPR(A)- and hPR(B)-mediated trans-activa tion. Unlike 8-Br-cAMP, phorbol 12-myristate 13-acetate did not cause marked ligand-independent trans-activation through either of the two r eceptor forms. RU486, an antagonist of progestin, preferentially block ed R5020-induced trans-activation compared to R5020 + 8-Br-cAMP synerg ism. As expected, H-89, a specific inhibitor of protein kinase A was m ore effective in inhibiting ligand-independent activity. Western analy sis of transfected receptors suggested that 8-Br-cAMP and 8-Br-cAMP R5020 but not R5020 alone down-regulated the level of hPR(B) in COS-1 cells. Only marginal modulation of hPR(A) levels was observed with R50 20 treatment in the presence and absence of 8-Br-cAMP. These data sugg est that R5020 and 8-Br-cAMP mediate PR-dependent transactivation thro ugh distinct pathways, and that phosphorylation can differentially reg ulate the activity of hPR(A) and hPR(B) formS, an observation which ma y be important for selective target gene activation in vivo by progest ins.