Synergistic enhancement of PRB-mediated RU486 and R5020 agonist activities through cyclic adenosine 3',5'-monophosphate represents a delayed primary response.

Abstract

Activators of protein kinase A have been shown to affect the transactivation potential of progestins and antiprogestins. To analyze the mechanisms and factors involved, we have created HeLa and CV1 cell clones stably expressing isoform B of progesterone receptor. In the HeLa cell background, the progesterone antagonist RU486 significantly induces progesterone-regulatable reporter genes, and this agonistic effect is synergistically enhanced by elevating cAMP or through overexpression of protein kinase A catalytic subunit. In contrast, in CV1 cells containing functional progesterone receptors no agonist activity of RU486 could be detected, suggesting the involvement of cell specifically expressed factors. In a PR(B)-positive HeLa cell clone containing stably integrated copies of a thymidine kinase-luciferase reporter gene with two progesterone response elements, we observed a complete loss of RU486 antagonist potential upon cotreatment with cAMP for 25 h while partial antagonist potential was maintained in a 5-h experiment. This result shows that, particularly in the presence of protein kinase A activators, the duration of hormone treatment is a crucial parameter in the evaluation of antagonist properties of antiprogestins. A detailed analysis of the kinetics of the hormone effects on transcription revealed that the onset of cAMP/RU486 synergism is delayed relative to the responses induced by RU486 or R5020 alone. Moreover, partial inhibition of protein synthesis by cycloheximide completely abolished cAMP/RU486 synergism while R5020 and RU486 responses were not inhibited. Together, these data indicate that cAMP/RU486 synergism is a delayed primary response requiring the intermediate induction of an essential factor.