Forskolin Stimulates Estrogen Receptor (ER) α Transcriptional Activity and Protects ER from Degradation by Distinct Mechanisms.

Estradiol action is mediated by estrogen receptors (ERs), a and ß . Estradiol binding initiates ER-mediated transcription and ER degradation, the latter of which occurs via the ubiquitin-proteasome pathway. Inhibition of proteasome activity prevents estradiol-induced ER α degradation and transactivation. In ER-positive GH3 cells (a rat pituitary prolactinoma cell line), forskolin, acting via protein kinase A (PKA), stimulates ER α transcriptional activity without causing degradation, and proteasome inhibition does not block forskolin-stimulated transcription. Forskolin also protects liganded ER α from degradation. In the current study, we first examined ER α and ER β transcriptional activity in ER-negative HT22 cells and found that forskolin stimulated ER α -, but not ER β -dependent transcription, through the ligand-binding domain (LBD). We also identified four mutations (L396R, D431Y, Y542F, and K534E/M548V) on the ER α LBD that selectively obliterated the response to forskolin. In GH3 cells, transfected ER α mutants and ER β were protected from degradation by forskolin. Ubiquitination of ER α and ER β was increased by forskolin or estradiol. ER α ubiquitination was diminished by a mutated ubiquitin (K48R) that prevents elongation of polyubiquitin chains for targeting the proteasome. Increased ER α ubiquitination was not affected by the deletion of the A/B domain but significantly diminished in the F domain deletion mutant. Our results indicate distinct and novel mechanisms for forskolin stimulation of ER α transcriptional activity and protection from ligand-induced degradation. It also suggests a unique mechanism by which forskolin increases unliganded and liganded ER α and ER β ubiquitination but uncouples them from proteasome-mediated degradation regardless of their transcriptional responses to forskolin.